Antibodies for Tumor Gangliosides

ABSTRACT

High affinity antibodies were made to gangliosides expressed on tumor cells. The antibodies can be used analytically, diagnostically, therapeutically, and theranostically. The antibodies may be used to target cytotoxic reagents to tumor cells, thus minimizing full-body toxicity. The antibodies may also be used with out added cytotoxin. The antibodies may be detectably labeled or labelable for analytic and diagnostic purposes. The combination of specificity and affinity of the antibodies render them particularly useful.

This application claims the benefit of and incorporates by reference theentire contents of U.S. 61/416,096 filed Nov. 22, 2010, and U.S.61/419,317 filed Dec. 3, 2010.

The invention was made using funds from the United States government.Therefore, the U.S. government retains certain rights in the inventionaccording to the terms of National Institutes of Health grants 5P50CA108786, 5P50 NS20023, and R37 CA011898.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of anti-tumor antibodies. Inparticular, it relates to antibodies to gangliosides prominentlydisplayed on tumor cell surfaces.

BACKGROUND OF THE INVENTION

Gangliosides are sialic acid-containing glycosphingolipids highlyenriched in the vertebrate nervous system. Although their functions havenot been fully clarified, they are thought to mediate neural cell-cellrecognition and modulate intracellular signaling [1]. Gangliosides areimplicated in various neural disorders, primarily the autoimmuneneuropathies, but also in storage disorders, e.g. Tay-Sachs disease orSandhoff disease [2-4]. High-affinity IgG anti-ganglioside antibodiesmay be used to develop animal models of autoimmune neuropathies andprobe normal ganglioside functions. However, it has been difficult toproduce high-affinity IgG antibodies against major brain gangliosides,especially GM1, GD1a, and GT1b [5]. This unresponsiveness has beenattributed to poor immunogenicity, T-cell independence, and tolerance[5-7]. Advances in genetics provide a potential solution to thisproblem. Several studies have shown that mice genetically engineered tolack the glycosyltransferase gene for ganglioside synthesis do notexpress complex gangliosides [8-12]. These mice, immunologically naiveto complex gangliosides, have been used for raising new antibodiesagainst complex gangliosides [2,6,13,14].

The Lc3-synthase gene (β1,3-N-acetylglucosaminyltransferase-V: β3Gn-T5)enzyme initiates the formation of the lacto-/neolacto-series gangliosideby transferring GlcNAc in a β1,3-linkage to lactosylceramide (FIG. 1A)[15]. The β3Gn-T5 is detected in mouse development and then again latermainly in the spleen and placenta in adult mice. Additionally,Lc3-synthase transcripts are found in cerebellar Purkinje cells of theadult mouse brain. On the other hand, lacto-series gangliosides such as3′-isoLM1 and 3′,6′-isoLD1 have been reported to be major mono- andoligo-sialogangliosides, respectively, of human gliomas [16-18]. Inthose studies, monoclonal antibodies (mAbs) such as SL-50, DMab-14, orDMab-22 recognizing lacto-series gangliosides were successfullyproduced; however, those antibodies proved to be of the low-affinity IgMsubclass.

There is a continuing need in the art to obtain high-specificity andhigh-affinity reagents for treating tumors, especially brain tumors.

SUMMARY OF THE INVENTION

According to one aspect of the invention a monoclonal antibody isprovided which has a higher affinity for 3′, 6′-isoLD1 ganglioside thanfor 3′ isoLM1 ganglioside. Additionally, it does not specifically bindto a ganglioside selected from the group consisting of isoLA1,Fuc3′-isoLM1, 3′-LM1, 3′, 8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2, GD1b,and GT1b.

Another aspect of the invention is an IgG monoclonal antibody whichbinds to one or more lacto-series gangliosides.

Yet another aspect of the invention is an isolated antibody constructwhich comprises a V_(H)CDR2 according to SEQ ID NO:1 and a V_(L)CDR1according to SEQ ID NO:2. The antibody construct binds to both 3′-isoLM1and 3′,6′-iso-LD1 gangliosides. The two CDRs can be in the same moleculeor in two subunits that heterodimerize.

Yet another aspect of the invention is a hybridoma cell which produces amonoclonal antibody which has a higher affinity for 3′,6′-isoLD1ganglioside than for 3′ isoLM1 ganglioside; additionally, it does notspecifically bind to a ganglioside selected from the group consisting ofisoLA1, Fuc3′-isoLM1, 3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2,GD1b, and GT1b. Alternatively, the monoclonal antibody is an IgGmonoclonal antibody which binds to one or more lacto-seriesgangliosides.

Still another aspect of the invention is a method of making a monoclonalantibody. Hybridoma cells are grown in culture. The hybridoma cellsproduce a monoclonal antibody which has a higher affinity for3′,6′-isoLD1 ganglioside than for 3′ isoLM1 ganglioside; additionally,it does not specifically bind to a ganglioside selected from the groupconsisting of isoLA1, Fuc3′-isoLM1, 3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2,GM1, GD3, GD2, GD1b, and GT1b. Alternatively, the hybridoma cellsproduce an antibody that is an IgG monoclonal antibody which binds toone or more lacto-series gangliosides. Monoclonal antibody is harvestedfrom the culture medium or from the hybridoma cells.

Another aspect of the invention is a method of detecting tumor cells ina tissue. A tissue sample is contacted with a monoclonal antibody whichhas a higher affinity for 3′,6′-isoLD1 ganglioside than for 3′ isoLM1ganglioside; additionally, it does not specifically bind to aganglioside selected from the group consisting of isoLA1, Fuc3′-isoLM1,3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2, GD1b, and GT1b. Or thetissue sample is contacted with a monoclonal antibody which is an IgGmonoclonal antibody which binds to one or more lacto-seriesgangliosides. Or the tissue sample is contacted with an isolatedantibody construct which comprises a V_(H)CDR2 according to SEQ ID NO:1and a V_(L)CDR1 according to SEQ ID NO:2. The presence of antibody orantibody construct bound to the tissue sample is detected.

Still another aspect of the invention is a method of treating a tumor ina human. An antibody or antibody construct is administered to the human.The antibody has a higher affinity for 3′,6′-isoLD1 ganglioside than for3′ isoLM1 ganglioside; additionally, it does not specifically bind to aganglioside selected from the group consisting of isoLA1, Fuc3′-isoLM1,3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2, GD1b, and GT1b, or theantibody is an IgG monoclonal antibody which binds to one or morelacto-series gangliosides, or the antibody is a construct whichcomprises a V_(H)CDR2 according to SEQ ID NO:1 and a V_(L)CDR1 accordingto SEQ ID NO:2. The monoclonal antibody or antibody construct can beattached to a cytotoxic agent.

Another aspect of the invention is an isolated nucleic acid moleculewhich encodes an scFv molecule comprising a V_(H)CDR2 according to SEQID NO:1 and a V_(L)CDR1 according to SEQ ID NO:2. The scFv binds to both3′-isoLM1 and 3′,6′-iso-LD1 gangliosides.

An aspect of the invention is a host cell comprising the isolatednucleic acid which encodes an scFv molecule comprising a V_(H)CDR2according to SEQ ID NO:1 and a V_(L)CDR1 according to SEQ ID NO:2. ThescFv binds to both 3′-isoLM1 and 3′,6′-iso-LD1 gangliosides.

A further aspect of the invention is a method of making an antibodyconstruct. The host cell comprising the isolated nucleic acid whichencodes an scFv molecule comprising a V_(H)CDR2 according to SEQ ID NO:1and a V_(L)CDR1 according to SEQ ID NO:2, wherein the scFv binds to both3′-isoLM1 and 3′,6′-iso-LD1 gangliosides is grown in culture medium. Theantibody construct is harvested from the culture medium or the hostcells.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with new reagentsfor treating and directing treatments to tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C. Production of an anti-3′-isoLM1/3′, 6′-isoLD1 gangliosideantibody. (FIG. 1A) Biosynthesis of lacto-/neolacto-series ganglioside.(FIG. 1B) Electrophoresis of 2 μg of GMab-1 under reducing conditions on4-10% NuPAGE gel. (FIG. 1C) ELISA of GMab-1 against 3′-isoLM1. The3′-isoLM1 conjugated with BSA was immobilized. After blocking, theplates were incubated with GMab-1 and isotype control at severalconcentrations.

FIG. 2A-2B. GMab-1 recognition of lacto-series ganglioside on the cellsurface of glioblastoma cells by flow cytometry. D54 cell line (FIG. 2A)or D54 xenograft cells (FIG. 2B), which were collected by 0.6 mM EDTAtreatment, were incubated with GMab-1 or isotype control.

FIG. 3A-3D. Epitope determination of GMab-1 in ELISA. Fifteengangliosides conjugated with BSA were immobilized. After blocking, theplates were incubated with GMab-1 (FIG. 3A), DMab-14 (FIG. 3B), SL-50(FIG. 3C), and DMab-22 (FIG. 3D) at a concentration of 10 μg/ml.

FIG. 4A-4D. Immunohistochemical analysis by GMab-1 against glioblastomatissues. Glioblastoma tissues were stained by GMab-1 (FIG. 4A, FIG. 4C)or isotype control (FIG. 4B, FIG. 4D). Magnification: ×200.

FIG. 5. Sequence alignment of DMab14-scFv (SEQ ID NOS: 9, 7, 10, 8) andDMab14-86184 (SEQ ID NOs: 3, 1, 4, 2) in VHCDR2 (SEQ ID NOS: 10, 8, 4,2, top to bottom) and VLCDR1 (SEQ ID NOS: 9, 7, 3, 1, top to bottom)regions. Differences are noted.

FIG. 6. DMab14-86184-scFv (VH-Linker-VL construction) nucleotide (SEQ IDNO:5) and amino acid (SEQ ID NO:6) sequence, G4S×3 linker at amino acidresidues approximately 161-175.

FIG. 7A-7B. DMab14-86184-scFv-PEKDEL protein binding affinity wasmeasured on D54MG cells by flow cytometry.

FIG. 8A-8B. DMab14-86184-scFv-PEKDEL protein binding affinity wasmeasured on H336 cells by flow cytometry.

FIG. 9A-9B. Cytotoxicity of DMab14-86184-scFv-PEKDEL protein on bothprotein synthesis inhibition assay (IC₅₀=80 ng/ml) and cellproliferation assay (IC₅₀<1000 ng/ml) against D54 MG cells.

FIG. 9C-9D. Cytotoxicity of DMab14-86184-scFv-PEKDEL on both proteinsynthesis inhibition assay (IC₅₀=4 ng/ml) and cell proliferation assay(IC₅₀-15 ng/ml) against H336 cells.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed antibodies (monoclonal or scFv construct)that have unique epitopic specificity for gangliosides and which havehigh affinity. The ganglioside targets of the antibody are found on thecell surfaces of certain tumors. This renders the antibodies usefulreagents for treating tumors and for delivering anti-tumor reagents totumors. Additionally the antibodies can be used analytically,prophylactically, prognostically, diagnostically, therapeutically, andtheranostically.

The antibodies of the invention may be produced using hybridoma cells.Hybridoma cells can be cultured and the monoclonal antibodies can beharvested from the culture medium or from the hybridoma cells.

One epitope to which the antibodies may bind is NeuAcα2-3Galβ1-3GlcNAc.The antibodies may bind with a higher affinity constant to 3′, 6′-isoLD1ganglioside than to 3′-isoLM1 ganglioside. The affinity constant for3′-isoLM1 ganglioside may be at least 1×10⁷, at least 5×10⁷, at least10⁸, or at least 5×10⁸ (mol/L)⁻¹. The affinity constant for 3′,6′-isoLD1 ganglioside will accordingly be higher. The antibodies may notbind to one or more of gangliosides isoLA1, Fuc3′-isoLM1, 3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2, GD1b, and GT1b. The antibodies maynot bind to any of these gangliosides. Non-binding may be less than0.05, less than 0.04, less than 0.03, less than 0.02 of the amount ofbinding to 3′-isoLM1 ganglioside.

The isotype of the antibodies is IgG. It may be any of IgG1, IgG2, IgG3,or IgG4 subtypes. Other types of antibodies may also be used if they canachieve sufficient binding affinities as shown here. Typically suchbinding affinities are at least 1×10⁷, at least 5×10⁷, at least 10⁸, orat least 5×10⁸ (mol/L)⁻¹ for 3′-isoLM1 ganglioside. In some cases othertypes of antibodies can be used and they can be “matured” to increasetheir affinity. Such maturation typically involves rounds of selectionfor binding-beneficial mutations. Other strategies for maturation can beused, including designing changes, rather than selecting among randomchanges.

Antibodies can be purified by methods well known in the art. Forexample, antibodies can be affinity purified by passage over a column towhich the relevant antigen is bound. The bound antibodies can then beeluted from the column using a buffer with a high salt concentration.

Hybridomas which produce desired anti-ganglioside antibodies can bepropagated in vitro as is known in the art to provide a long-lastingsource of antibodies. Alternatively, hybridoma cells can be injectedintraperitoneally into mice, which will then produce tumors. Thesetumors are accompanied by the production of ascites fluid which containsthe desired monoclonal antibodies. The monoclonal antibodies can berecovered from the ascites fluid by conventional methods such asultrafiltration, ultracentrifugation, dialysis, and immunoaffinitychromatography.

Single-chain antibodies can be constructed, for example, using hybridomacDNA as a template and a DNA amplification method, such as PCR (Thirionet al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodiescan be mono- or bispecific, and can be bivalent or tetravalent.Construction of tetravalent, bispecific single-chain antibodies istaught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15,159-63. Construction of bivalent, bispecific single-chain antibodies istaught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206. As anon-limiting example of such a construct see SEQ ID NO:5 and 6.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91).

The presence of antibody (indicating the presence of analyte) can bedetected, analytically, in a body sample, such as a tissue or bodyfluid, for example. The antibody can be labeled before contacting with asample, for example, with a radionuclide or an enzyme. Alternatively theantibody is initially unlabeled, and after contacting it is labeled byuse of a second antibody which recognizes the type of the firstantibody. The second antibody may be detectably labeled. Such detectionsystems are well known in the art. The format of the assay can be anythat is known in the art. The antibody can be used inimmunohistochemistry, or in an ELISA format, for example. The antibodycan be used in a fluorescence activated cell sorter. Many differenttechniques and formats for detecting bound antibody are known and can beused. Antibodies can be attached to solid supports, such as wells,arrays, beads, and microspheres.

Any toxic moiety can be attached to the antibodies to achieve deliveryof the toxic moiety to the appropriate site in the body, typically thesite of the tumor. One such toxin which can be used is Pseudomonasexotoxin (PE38). Other suitable therapeutic agents include smallmolecule cytotoxic agents, i.e., compounds with the ability to killmammalian cells and having a molecular weight of less than 700 daltons.Such compounds may also contain toxic metals capable of having acytotoxic effect. Such small molecule cytotoxic agents includepro-drugs, i.e., compounds that decay or are converted underphysiological conditions to release cytotoxic agents. Examples of suchagents include cisplatin, maytansine derivatives, rachelmycin,calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide,irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II,temozolmide, topotecan, trimetreate glucuronate, auristatin Evincristine and doxorubicin. Other toxic moieties which may be used arepeptide cytotoxins, i.e., proteins or fragments of proteins that havethe ability to kill mammalian cells. Examples include ricin, diphtheriatoxin, Pseudomonas bacterial exotoxin A, DNAase and RNAase. Additionaltoxic moieties that may be used are radionuclides, i.e., unstableisotopes of elements which decay thereby emitting one or more of α or βparticles, or γ rays. Examples include iodine 131, rhenium 186, indium111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213.Means for attaching, conjugating, binding, complexing, and chelatingtoxic moieties to antibodies are well known in the art. A method that isappropriate for the agent and the use of the antibody can be chosenreadily by the skilled artisan.

The antibodies are useful for treating, alone or as part of acombination treatment, a variety of tumors. These include but are notlimited to brain tumors such as glioblastoma, breast, prostate, colon,renal cell, pancreas, melanoma, seminoma, germinoma, teratoma, andleiomyosarcoma. Not all tumors may express the targeted gangliosides.Tumor cells which do express the targeted gangliosides on their surfacesare likely more susceptible than those which do not. Thus one may wantto pre-screen patients to be treated by testing their tumor cells forexpression, and preferably robust expression, of the targetedgangliosides. Thus, a treatment can be personalized and unnecessarytreatments can be avoided. Thus unnecessary expense and possible sideeffects can be avoided.

DEFINITIONS

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

The term “ganglioside” refers to those sialylated glycosphingolipidswhich constitute the major class of glycoconjugates on neurons and carrythe majority of the sialic acid within the central nervous system.Gangliosides, a group of the glioma-associated antigens, are believed toplay a role in tumor formation and significantly impact tumorprogression. They are involved in various cellular functions, includingsignal transduction, regulation of cell proliferation anddifferentiation, cell-cell regulation and adhesion, and cell death (see,e.g., Hwang, L. et al. 2010). In preferred embodiments, the term“gangliosides” refers to 3′iso-LM1 and/or 3′6′iso-LD1, both of whichhave been characterized and validated as molecular targets for thetreatment of malignant gliomas because of their overexpression in thosetumors, especially glioblastoma multiforme (see, e.g., Wikstrand,Fredman et al., 1992; Fredman 1994).

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies), heteroconjugate antibodies (e.g.,bispecific antibodies) and recombinant single chain Fv fragments (scFv),disulfide stabilized (dsFv) Fv fragments (See, U.S. Ser. No. 08/077,252,incorporated herein by reference), or pFv fragments (See, U.S.Provisional Patent Applications 60/042,350 and 60/048,848, both of whichare incorporated herein by reference.). The term “antibody” alsoincludes antigen binding forms of antibodies (e.g., Fab′, F(ab′)₂, Fab,Fv and rIgG (See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.).

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors (See, e.g., Huse, etal., Science 246:1275-1281 (1989); Ward, et al., Nature 341:544-546(1989); and Vaughan, et al., Nature Biotech. 14:309-314 (1996)).

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region. Light andheavy chain variable regions contain a “framework” region interrupted bythree hypervariable regions, also called complementarity-determiningregions or CDRs. The extent of the framework region and CDRs have beendefined (see, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Kabat,E., et al., U.S. Department of Health and Human Services, (1987); whichis incorporated herein by reference). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three dimensional space. TheCDRs are primarily responsible for binding to an epitope of an antigen.The CDRs are typically referred to as CDR1, CDR2, and CDR3, numberedsequentially starting from the N-terminus.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe heavy chain and the light chain of a traditional two chain antibodyhave been joined to form one chain. Typically, a linker peptide isinserted between the two chains to allow for proper folding and creationof an active binding site.

The term “linker peptide” includes reference to a peptide within anantibody binding fragment (e.g., Fv fragment) which serves to indirectlybond the variable heavy chain to the variable light chain.

The term “contacting” includes reference to placement in direct physicalassociation. With regards to this invention, the term refers toantibody-antigen binding.

As used herein, the term “anti-ganglioside” in reference to an antibody,includes reference to an antibody which is generated againstganglioside, such as 3′isoLM1 and 3′6′isoLD1. In certain embodiments,the ganglioside is a primate ganglioside such as human ganglioside. Inother embodiments, the antibody is generated against human gangliosidesynthesized by a non-primate mammal after introduction into the animalof cDNA which encodes human ganglioside.

As used herein, “polypeptide”, “peptide” and “protein” are usedinterchangeably and include reference to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The terms also apply to polymers containing conservativeamino acid substitutions such that the protein remains functional.

The term “residue” or “amino acid residue” or “amino acid” includesreference to an amino acid that is incorporated into a protein,polypeptide, or peptide (collectively “peptide”). The amino acid can bea naturally occurring amino acid and, unless otherwise limited, canencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

The phrase “disulfide bond” or “cysteine-cysteine disulfide bond” refersto a covalent interaction between two cysteines in which the sulfuratoms of the cysteines are oxidized to form a disulfide bond. Theaverage bond energy of a disulfide bond is about 60 kcal/mol compared to1-2 kcal/mol for a hydrogen bond. In the context of this invention, thecysteines which form the disulfide bond are within the framework regionsof the single chain antibody and serve to stabilize the conformation ofthe antibody.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule. In thecontext of the present invention, the terms include reference to joiningan antibody moiety to an effector molecule (EM). The linkage can beeither by chemical or recombinant means. Chemical means refers to areaction between the antibody moiety and the effector molecule such thatthere is a covalent bond formed between the two molecules to form onemolecule.

As used herein, “recombinant” includes reference to a protein producedusing cells that do not have, in their native state, an endogenous copyof the DNA able to express the protein. The cells produce therecombinant protein because they have been genetically altered by theintroduction of the appropriate isolated nucleic acid sequence. The termalso includes reference to a cell, or nucleic acid, or vector, that hasbeen modified by the introduction of a heterologous nucleic acid or thealteration of a native nucleic acid to a form not native to that cell,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell, express mutants of genes that arefound within the native form, or express native genes that are otherwiseabnormally expressed, under expressed or not expressed at all.

As used herein, “nucleic acid” or “nucleic acid sequence” includesreference to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence includesthe complementary sequence thereof as well as conservative variants,i.e., nucleic acids present in wobble positions of codons and variantsthat, when translated into a protein, result in a conservativesubstitution of an amino acid.

As used herein, “encoding” with respect to a specified nucleic acid,includes reference to nucleic acids which comprise the information fortranslation into the specified protein. The information is specified bythe use of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum (Proc. Nat'l Acad.Sci. USA 82:2306-2309 (1985), or the ciliate Macronucleus, may be usedwhen the nucleic acid is expressed in using the translational machineryof these organisms.

The phrase “fusing in frame” refers to joining two or more nucleic acidsequences which encode polypeptides so that the joined nucleic acidsequence translates into a single chain protein which comprises theoriginal polypeptide chains.

As used herein, “expressed” includes reference to translation of anucleic acid into a protein. Proteins may be expressed and remainintracellular, become a component of the cell surface membrane or besecreted into the extracellular matrix or medium.

By “host cell” is meant a cell which can support the replication orexpression of the expression vector. Host cells may be prokaryotic cellssuch as E. coli, or eukaryotic cells such as yeast, insect, amphibian,or mammalian cells.

The phrase “phage display library” refers to a population ofbacteriophage, each of which contains a foreign cDNA recombinantly fusedin frame to a surface protein. The phage displays the foreign proteinencoded by the cDNA on its surface. After replication in a bacterialhost, typically E. coli, the phage which contain the foreign cDNA ofinterest are selected by the expression of the foreign protein on thephage surface.

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” include reference to a dosage of atherapeutic agent sufficient to produce a desired result, such asinhibiting cell protein synthesis by at least 50%, or killing the cell.

The term “therapeutic agent” includes any number of compounds currentlyknown or later developed to act as anti-neoplastics,anti-inflammatories, cytokines, anti-infectives, enzyme activators orinhibitors, allosteric modifiers, antibiotics or other agentsadministered to induce a desired therapeutic effect in a patient.

The term “immunoconjugate” includes reference to a covalent linkage ofan effector molecule to an antibody. The effector molecule can be animmunotoxin.

The term “toxin” includes reference to abrin, ricin, Pseudomonasexotoxin (PE), diphtheria toxin (DT), botulinum toxin, or modifiedtoxins thereof. For example, PE and DT are highly toxic compounds thattypically bring about death through liver toxicity. PE and DT, however,can be modified into a form for use as an immunotoxin by removing thenative targeting component of the toxin (e.g., domain Ia of PE and the Bchain of DT) and replacing it with a different targeting moiety, such asan antibody.

The term “in vivo” includes reference to inside the body of the organismfrom which the cell was obtained. “Ex vivo” and “in vitro” means outsidethe body of the organism from which the cell was obtained.

The phrase “malignant cell” or “malignancy” refers to tumors or tumorcells that are invasive and/or able to undergo metastasis, i.e., acancerous cell.

As used herein, “mammalian cells” includes reference to cells derivedfrom mammals including humans, rats, mice, guinea pigs, chimpanzees, ormacaques. The cells may be cultured in vivo or in vitro.

The term “selectively reactive” includes reference to the preferentialassociation of an antibody, in whole or part, with a cell or tissuebearing gangliosides and not to cells or tissues lacking gangliosides.It is, of course, recognized that a certain degree of non-specificinteraction may occur between a molecule and a non-target cell ortissue. Nevertheless, selective reactivity, may be distinguished asmediated through specific recognition of gangliosides. Althoughselectively reactive antibodies bind antigen, they may do so with lowaffinity. On the other hand, specific binding results in a much strongerassociation between the antibody and cells bearing gangliosides thanbetween the bound antibody and cells lacking gangliosides or lowaffinity antibody-antigen binding.

Specific binding typically results in greater than 2-fold, preferablygreater than 5-fold, more preferably greater than 10-fold and mostpreferably greater than 100-fold increase in amount of bound antibody(per unit time) to a cell or tissue bearing gangliosides as compared toa cell or tissue lacking gangliosides. Specific binding to a proteinunder such conditions requires an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsare appropriate for selecting antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

The term “immunologically reactive conditions” includes reference toconditions which allow an antibody generated to a particular epitope tobind to that epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. Preferably, the immunologically reactive conditions employedin the methods of the present disclosure are “physiological conditions”which include reference to conditions (e.g., temperature, osmolarity,pH) that are typical inside a living mammal or a mammalian cell. Whileit is recognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment normally lies around pH 7(i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above 0°C. and below 50° C. Osmolarity is within the range that is supportive ofcell viability and proliferation.

The anti-ganglioside antibodies employed in the present disclosure canbe linked to effector molecules (EM) through the EM carboxyl terminus,the EM amino terminus, through an interior amino acid residue of the EMsuch as cysteine, or any combination thereof. Similarly, the EM can belinked directly to the heavy, light, Fc (constant region) or frameworkregions of the antibody. Linkage can occur through the antibody's aminoor carboxyl termini, or through an interior amino acid residue. Further,multiple EM molecules (e.g., any one of from 2-10) can be linked to theanti-ganglioside antibody and/or multiple antibodies (e.g., any one offrom 2-5) can be linked to an EM. The antibodies used in a multivalentimmunoconjugate composition of the present invention can be directed tothe same or different ganglioside epitopes.

In certain embodiments of the present disclosure, the anti-gangliosideantibody is a recombinant antibody such as a scFv or disulfidestabilized Fv antibody. Fv antibodies are typically about 25 kDa andcontain a complete antigen-binding site with 3 CDRs per heavy and lightchain. If the V_(H) and the V_(L) chain are expressed non-contiguously,the chains of the Fv antibody are typically held together by noncovalentinteractions. However, these chains tend to dissociate upon dilution, somethods have been developed to crosslink the chains throughglutaraldehyde, intermolecular disulfides, or a peptide linker.

In other embodiments, the antibody is a single chain Fv (scFv). TheV_(H) and the V_(L) regions of a scFv antibody comprise a single chainwhich is folded to create an antigen binding site similar to that foundin two-chain antibodies. Once folded, noncovalent interactions stabilizethe single chain antibody. In certain embodiments, the scFv isrecombinantly produced. One of skill will realize that conservativevariants of the antibodies of the instant invention can be made. Suchconservative variants employed in scFv fragments will retain criticalamino acid residues necessary for correct folding and stabilizingbetween the V_(H) and the V_(L) regions. Conservatively modifiedvariants of the prototype sequence have at least 80% sequencesimilarity, preferably at least 85% sequence similarity, more preferablyat least 90% sequence similarity, and most preferably at least 95%sequence similarity at the amino acid level to its prototype sequence.

In some embodiments of the present invention, the scFv antibody isdirectly linked to the EM through the light chain. However, scFvantibodies can be linked to the EM via its amino or carboxyl terminus.

While the V_(H) and V_(L) regions of some antibody embodiments can bedirectly joined together, one of skill will appreciate that the regionsmay be separated by a peptide linker consisting of one or more aminoacids. Peptide linkers and their use are well-known in the art. See,e.g., Huston, et al., Proc. Nat'l Acad. Sci. USA 8:5879 (1988); Bird, etal., Science 242:4236 (1988); Glockshuber, et al., Biochemistry 29:1362(1990); U.S. Pat. No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer, etal., Biotechniques 14:256-265 (1993), all incorporated herein byreference. Generally the peptide linker will have no specific biologicalactivity other than to join the regions or to preserve some minimumdistance or other spatial relationship between them. However, theconstituent amino acids of the peptide linker may be selected toinfluence some property of the molecule such as the folding, net charge,or hydrophobicity. Single chain Fv (scFv) antibodies optionally includea peptide linker of no more than 50 amino acids, generally no more than40 amino acids, preferably no more than 30 amino acids, and morepreferably no more than 20 amino acids in length. However, it is to beappreciated that some amino acid substitutions within the linker can bemade. For example, a valine can be substituted for a glycine.

Antibody Production

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably isolated ganglioside orextracellular ganglioside epitopes are mixed with an adjuvant andanimals are immunized with the mixture. When appropriately high titersof antibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. If desired, further fractionation ofthe antisera to enrich for antibodies reactive to the polypeptide isperformed. See, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY,Wiley/Greene, NY (1991); and Harlow & Lane, supra, which areincorporated herein by reference.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) BASICAND CLINICAL IMMUNOLOGY (4TH ED.), Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow & Lane, supra;Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2D ED.),Academic Press, New York, N.Y. (1986); Kohler & Milstein, Nature256:495-497 (1975); and particularly (Chowdhury, P. S., et al., Mol.Immunol. 34:9 (1997)), which discusses one method of generatingmonoclonal antibodies.

Binding Affinity of Antibodies

The antibodies of this disclosure specifically bind to an extracellularepitope of ganglioside. An anti-ganglioside antibody has bindingaffinity for ganglioside if the antibody binds or is capable of bindingganglioside as measured or determined by standard antibody-antigenassays, for example, competitive assays, saturation assays, or standardimmunoassays such as ELISA or RIA.

Such assays can be used to determine the dissociation constant of theantibody. The phrase “dissociation constant” refers to the affinity ofan antibody for an antigen. Specificity of binding between an antibodyand an antigen exists if the dissociation constant (k_(D))=1/K, where Kis the affinity constant) of the antibody is <1 μM, preferably <100 nM,and most preferably <0.1 nM. Antibody molecules will typically have ak_(D) in the lower ranges. k_(D)=[Ab-Ag]/[Ab][Ag] where [Ab] is theconcentration at equilibrium of the antibody, [Ag] is the concentrationat equilibrium of the antigen and [Ab-Ag] is the concentration atequilibrium of the antibody-antigen complex. Typically, the bindinginteractions between antigen and antibody include reversible noncovalentassociations such as electrostatic attraction, Van der Waals forces andhydrogen bonds. This method of defining binding specificity applies tosingle heavy and/or light chains, CDRs, fusion proteins or fragments ofheavy and/or light chains, that are specific for ganglioside if theybind ganglioside alone or in combination.

Production of Immunoconjugates

Immunoconjugates include, but are not limited to, molecules in whichthere is a covalent linkage of a therapeutic agent to an antibody. Atherapeutic agent is an agent with a particular biological activitydirected against a particular target molecule or a cell bearing a targetmolecule. One of skill in the art will appreciate that therapeuticagents may include various drugs such as vinblastine, daunomycin and thelike, cytotoxins such as native or modified Pseudomonas exotoxin orDiphtheria toxin, encapsulating agents, (e.g., liposomes) whichthemselves contain pharmacological compositions, radioactive agents suchas ¹²⁵I, ³²P, ¹⁴C, ³H, and ³⁵S and other labels, target moieties andligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell and the biological effect is desired to evoke.Thus, for example, the therapeutic agent may be a cytotoxin which isused to bring about the death of a particular target cell. Conversely,where it is merely desired to invoke a non-lethal biological response,the therapeutic agent may be conjugated to a non-lethal pharmacologicalagent or a liposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies herein provided, one of skillcan readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same EM or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusion proteins thereof.

Pseudomonas Exotoxin and Other Toxins

Toxins can be employed with antibodies of the present disclosure toyield immunotoxins. Exemplary toxins include ricin, abrin, diphtheriatoxin and subunits thereof, as well as botulinum toxins A through F.These toxins are readily available from commercial sources (e.g., SigmaChemical Company, St. Louis, Mo.). Diptheria toxin is isolated fromCorynebacterium diphtheriae. Ricin is the lectin RCA60 from Ricinuscommunis (Castor bean). The term also references toxic variants thereof.For example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401. Ricinuscommunis agglutinin (RCA) occurs in two forms designated RCA.sub.60 andRCA.sub.120 according to their molecular weights of approximately 65 and120 k_(D) respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta266:543 (1972)). The A chain is responsible for inactivating proteinsynthesis and killing cells. The B chain binds ricin to cell-surfacegalactose residues and facilitates transport of the A chain into thecytosol (Olsnes, et al., Nature 249:627-631 (1974) and U.S. Pat. No.3,060,165).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 k_(D) and are composed of two disulfide-linked polypeptidechains A and B. The A chain inhibits protein synthesis; the B-chain(abrin-b) binds to D-galactose residues (see, Funatsu, et al., Agr.Biol. Chem. 52:1095 (1988); and Olsnes, Methods Enzymol. 50:330-335(1978)).

In preferred embodiments of the present disclosure, the toxin isPseudomonas exotoxin (PE). The term “Pseudomonas exotoxin” as usedherein refers to a full-length native (naturally occurring) PE or a PEthat has been modified. Such modifications may include, but are notlimited to, elimination of domain Ia, various amino acid deletions indomains Ib, II and III, single amino acid substitutions and the additionof one or more sequences at the carboxyl terminus such as KDEL and REDL.See Siegall, et al., J. Biol. Chem. 264:14256 (1989). In a preferredembodiment, the cytotoxic fragment of PE retains at least 50%,preferably 75%, more preferably at least 90%, and most preferably 95% ofthe cytotoxicity of native PE. In a most preferred embodiment, thecytotoxic fragment is more toxic than native PE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The native PEsequence is provided as SEQ ID NO:1 of U.S. Pat. No. 5,602,095,incorporated herein by reference. The method of action is inactivationof the ADP-ribosylation of elongation factor 2 (EF-2). The exotoxincontains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2. The function of domain Ib (aminoacids 365-399) remains undefined, although a large part of it, aminoacids 365-380, can be deleted without loss of cytotoxicity. See Siegall,et al., J. Biol. Chem. 264: 14256-14261 (1989), incorporated byreference herein.

PE employed in the present invention include the native sequence,cytotoxic fragments of the native sequence, and conservatively modifiedvariants of native PE and its cytotoxic fragments. Cytotoxic fragmentsof PE include those which are cytotoxic with or without subsequentproteolytic or other processing in the target cell (e.g., as a proteinor pre-protein). Cytotoxic fragments of PE include PE40, PE38, and PE35.PE40 is a truncated derivative of PE as previously described in the art.See, Pai, et al., Proc. Nat'l Acad. Sci. USA 88:3358-62 (1991); andKondo, et al., J. Biol. Chem. 263:9470-9475 (1988). PE35 is a 35 K_(D)carboxyl-terminal fragment of PE composed of a met at position 280followed by amino acids 281-364 and 381-613 of native PE. In otherembodiments, the cytotoxic fragment PE38 is employed. PE38 is atruncated PE pro-protein composed of amino acids 253-364 and 381-613which is activated to its cytotoxic form upon processing within a cell(see U.S. Pat. No. 5,608,039, incorporated herein by reference).

In certain embodiment, PE38 is the toxic moiety of the immunotoxin ofthis disclosure, however, other cytotoxic fragments PE35 and PE40 arecontemplated and are disclosed in U.S. Pat. Nos. 5,602,095 and4,892,827, each of which is incorporated herein by reference.

Other Therapeutic Moieties

Antibodies of the present disclosure can also be used to target anynumber of different diagnostic or therapeutic compounds to cellsexpressing ganglioside on their surface. Thus, an antibody of thepresent invention, such as an anti-ganglioside scFv, may be attacheddirectly or via a linker to a drug that is to be delivered directly tocells bearing ganglioside. Therapeutic agents include such compounds asnucleic acids, proteins, peptides, amino acids or derivatives,glycoproteins, radioisotopes, lipids, carbohydrates, or recombinantviruses. Nucleic acid therapeutic and diagnostic moieties includeantisense nucleic acids, derivatized oligonucleotides for covalentcross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-ganglioside antibody maybe an encapsulation system, such as a liposome or micelle that containsa therapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735;and Connor, et al., Pharm. Ther. 28:341-365 (1985).

Detectable Labels

Antibodies of the present disclosure may optionally be covalently ornoncovalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. DYNABEADS), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Conjugation to the Antibody

In a non-recombinant embodiment of the invention, effector molecules,e.g., therapeutic, diagnostic, or detection moieties, are linked to theanti-ganglioside antibodies of the present disclosure using any numberof means known to those of skill in the art. Both covalent andnoncovalent attachment means may be used with anti-gangliosideantibodies of the present disclosure.

The procedure for attaching an effector molecule to an antibody willvary according to the chemical structure of the EM. Polypeptidestypically contain variety of functional groups; e.g., carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule.

Alternatively, the antibody is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

A “linker”, as used herein, is a molecule that is used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (e.g.,through a disulfide linkage to cysteine). However, in certainembodiments, the linkers will be joined to the alpha carbon amino andcarboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages which are cleavable in the vicinity of the target site.Cleavage of the linker to release the effector molecule from theantibody may be prompted by enzymatic activity or conditions to whichthe immunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

Pharmaceutical Compositions and Administration

The antibody and/or immunoconjugate compositions of this disclosure(i.e., PE linked to an anti-ganglioside antibody), are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. For example,ovarian malignancies may be treated by intravenous administration or bylocalized delivery to the tissue surrounding the tumor.

The compositions for administration will commonly comprise a solution ofthe antibody and/or immunoconjugate dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical immunotoxin composition of the presentinvention for intravenous administration would be about 0.1 to 10 mg perpatient per day. Dosages from 0.1 up to about 100 mg per patient per daymay be used, particularly if the drug is administered to a secluded siteand not into the circulatory or lymph system, such as into a body cavityor into a lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as REMINGTON'SPHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa.(1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the immunoconjugatecompositions of the present disclosure can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μM so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed.,Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice &Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., MarcelDekker, Inc. New York, N.Y., pp. 315-339 (1992) both of which areincorporated herein by reference.

Polymers can be used for ion-controlled release of immunoconjugatecompositions of the present invention. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).For example, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent. Sci.Tech. 44(2):58-65 (1990)). Alternatively, hydroxyapatite has been usedas a microcarrier for controlled release of proteins (Ijntema, et al.,Int. J. Pharm. 112:215-224 (1994)). In yet another aspect, liposomes areused for controlled release as well as drug targeting of thelipid-capsulated drug (Betageri, et at., LIPOSOME DRUG DELIVERY SYSTEMS,Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerousadditional systems for controlled delivery of therapeutic proteins areknown. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871,4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670;5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961;5,254,342 and 5,534,496, each of which is incorporated herein byreference.

Among various uses of the immunotoxins of the present invention areincluded a variety of disease conditions caused by specific human cellsthat may be eliminated by the toxic action of the fusion protein. Onepreferred application for the immunotoxins of the invention is thetreatment of malignant cells expressing gangliosides. Exemplarymalignant cells include astrocytomas, glioblastomas, and the like.

Diagnostic Kits

In another embodiment, this invention provides for kits for thedetection of ganglioside or an immunoreactive fragment thereof, (i.e.,collectively, a “ganglioside protein”) in a biological sample. A“biological sample” as used herein is a sample of biological tissue orfluid that contains ganglioside. Such samples include, but are notlimited to, tissue from biopsy, sputum, amniotic fluid, blood, and bloodcells (e.g., white cells). Fluid samples may be of some interest, butare generally not preferred herein since detectable concentrations ofganglioside are rarely found in such a sample. Biological samples alsoinclude sections of tissues, such as frozen sections taken forhistological purposes. A biological sample is typically obtained from amulticellular eukaryote, preferably a mammal such as rat, mice, cow,dog, guinea pig, or rabbit, and most preferably a primate such asmacaques, chimpanzees, or humans.

Kits will typically comprise an anti-ganglioside antibody of the presentdisclosure. In some embodiments, the anti-ganglioside antibody will bean anti-ganglioside Fv fragment; preferably a scFv fragment.

In addition the kits will typically include instructional materialsdisclosing means of use of an antibody of the present disclosure (e.g.for detection of glioma cells in a sample). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. Thus, for example, the kit may additionally containmeans of detecting the label (e.g. enzyme substrates for enzymaticlabels, filter sets to detect fluorescent labels, appropriate secondarylabels such as a sheep anti-mouse-HRP, or the like). The kits mayadditionally include buffers and other reagents routinely used for thepractice of a particular method. Such kits and appropriate contents arewell known to those of skill in the art.

In one embodiment of the present disclosure, the diagnostic kitcomprises an immunoassay. As described above, although the details ofthe immunoassays of the present disclosure may vary with the particularformat employed, the method of detecting ganglioside in a biologicalsample generally comprises the steps of contacting the biological samplewith an antibody which specifically reacts, under immunologicallyreactive conditions, to ganglioside. The antibody is allowed to bind toganglioside under immunologically reactive conditions, and the presenceof the bound antibody is detected directly or indirectly.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1 Materials and Methods Animals, Cell Lines, Xenograft, andTissues

The β3Gn-T5 knockout mice were recently developed at Duke UniversityMedical Center. P3U1 cells were obtained from the American Type CultureCollection (Manassas, Va.), and we established a D54 glioblastoma cellline at Duke [16]. P3U1 and D54 cells were cultured at 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air in RPMI 1640 mediumincluding 2 mM L-glutamine (Invitrogen Corp., Carlsbad, Calif.) and 1%of penicillin-streptomycin solution (Invitrogen Corp.) or Zinc Optionmedium supplemented with 10% heat-inactivated fetal bovine serum (FBS;Sigma, St. Louis, Mo.), respectively. We established and maintained aD54 xenograft at Duke, using human tissue from anonymous donors, whichwas obtained from the Tissue Bank at the Preston Robert Tisch BrainTumor Center at Duke.

Antibodies and Gangliosides

Anti-ganglioside antibodies SL-50, DMab-14, and DMab-22 were previouslyproduced at the University of Gothenburg (SL-50) and at Duke University(DMab-14 and -22) [16-18]. Isotype control of mouse IgG₃ was purchasedfrom eBioscience, Inc. (San Diego, Calif.). All gangliosides used forimmunization or enzyme-linked immunosorbent assay (ELISA) were isolatedand characterized at the University of Gothenburg as describedpreviously [19].

Hybridoma Production

The β3Gn-T5 knockout mice were immunized by neck subcutaneous.injections of 20 μg of purified 3′-isoLM1 and 3′,6′-isoLD1 coupled toSalmonella minnesota with Imject Freund's Complete Adjuvant (ThermoScientific Inc., Rockford, Ill.). One week later, secondary i.p.immunization of 20 μg of purified gangliosides was performed. Afteradditional immunization of 20 μg of purified gangliosides, a boosterinjection was given i.p. 2 days before spleen cells were harvested. Thespleen cells were fused with mouse myeloma P3U1 cells by using Sendaivirus (hemagglutinating virus of Japan: HVJ) envelope: GenomONE-CF_(EX)(Cosmo Bio USA, Inc., Carlsbad, Calif.) according to the manufacturer'sinstructions. The hybridomas were grown in RPMI medium includinghypoxanthine, aminopterin, and thymidine selection medium supplement(Sigma), 2 mM L-glutamine (Invitrogen Corp.), 10% heat-inactivated FBS(Sigma), 5% BriClone (QED Bioscience Inc., San Diego, Calif.), and 1% ofpenicillin-streptomycin solution (Invitrogen Corp.). The culturesupernatants were screened by ELISA for the binding to 3′-isoLM1conjugated with bovine serum albumin (BSA). Single cell cloning wasperformed with ClonaCell-HY Hybridoma Selection Medium (Medium D;StemCell Technologies Inc., Vancouver, BC, Canada). The IgG subclass wasdetermined by IsoStrip Mouse Monoclonal Antibody Isotyping Kit (RocheDiagnostics Corp., Indianapolis, Ind.).

ELISA

For evaluation by ELISA, gangliosides conjugated with BSA wereimmobilized on 96-well plates at 1 μg/ml for 30 min. After blocking with1% BSA in PBS, the plates were incubated with primary antibodies atseveral concentrations, followed by 1:1000 diluted peroxidase-conjugatedanti-mouse IgG (GE Healthcare UK Ltd., Buckinghamshire, England). Theenzymatic reaction was conducted with a substrate solution containing3,3′,5,5′-tetramethylbenzidine (TMB; Thermo Scientific Inc.). After thereaction was stopped with 2 M H₂SO₄, the optical density was measured at450 nm with a microplate reader (Bio-Rad Laboratories, Inc.,Philadelphia, Pa.). These reactions were performed with a volume of 50μl at room temperature.

SDS-PAGE

For sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2 μg ofeach protein was electrophoresed under reducing conditions on 4-10%NuPAGE gel (Invitrogen). The gel was stained with Bio-Safe Coomassie(Bio-Rad Laboratories, Inc.).

Flow Cytometry

D54 or D54 xenograft cells, which were collected by 0.6 mM EDTAtreatment, were incubated with GMab-1 or isotype control (10 μg/ml) for1 h at 4° C. Then the cells were incubated with the Oregongreen-conjugated anti-mouse antibody (1/200 dilution; Invitrogen Corp.)for 30 min. Flow cytometry was performed with a FACS Calibur (BectonDickinson, Franklin Lakes, N.J.).

Affinity Constant Determination by Surface Plasmon Resonance

To determine the affinity constant (K_(A)), purified GMab-1 wasimmobilized on the surface of biosensor chips for analysis by using theBIAcore 3000 system (BIAcore, Piscataway, N.J.). Coupling of antigen wasachieved by using N-ethyl-N-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide according to the instructions of themanufacturer. The running buffer was 10 nM HEPES, 150 mM NaCl, and 3.4mM EDTA (pH 7.4). The gangliosides conjugated with BSA and control BSAwere passed over the biosensor chip and affinity rate constants(association rate constant: k_(assoc) and disassociation rate constant:k_(diss)) were determined by nonlinear curve-fitting using the Langmuirone-site binding model of the BIAevaluation software (BIAcore). K_(A) atequilibrium was calculated as K_(A)=k_(assoc)/k_(diss).

Immunohistochemical Analysis

All procedures were performed with the Ventana Discovery XT system(Ventana Medical Systems, Inc., Tucson, Ariz.). Briefly, acetone-fixed5- to 8-μm frozen tissue sections of human glioblastoma tissues wereincubated with GMab-1 (10 μg/ml) or isotype control (10 μg/ml) for 30min. The universal biotin-conjugated secondary antibody was incubatedfor 20 min followed by the peroxidase-conjugated streptavidin for 16min. Color was developed by using 3,3′-diaminobenzidinetetrahydrochloride for 8 min, and counterstained by hematoxylin.

Example 2 Production of an Anti-3′-isoLM1/3′,6′-isoLD1-Specific Antibody

Monoclonal antibodies recognizing lacto-series gangliosides such as3′-isoLM1 and 3′,6′-isoLD1 were previously produced and fullycharacterized [16,17]. The 3′-isoLM1 and 3′,6′-isoLD1 gangliosides areexpressed at high frequency in human glioblastomas and are rarelydetected in the normal adult brain. Therefore, 3′-isoLM1 and3′,6′-isoLD1 are ideal molecular targets for immunotherapy againstglioblastomas using specific mAbs. However, those established mAbs haverelatively low-affinity against 3′-isoLM1 and 3′,6′-isoLD1 because theyare of the IgM subclass. In several studies, anti-ganglio-seriesganglioside mAbs with high affinity have been developed by immunizingGalNAcT^(−/−) mice with ganglio-series gangliosides [2,6,13,14].Similarly, in this study, we immunized β3Gn-T5 knockout mice withpurified 3′-isoLM1 and 3′,6′-isoLD1 coupled to Salmonella minnesota togenerate high-affinity anti-lacto-series ganglioside mAbs (FIG. 1A).Because the hybridomas that we previously produced from β3Gn-T5 knockoutmice using the conventional polyethylene glycol fusion method had lowviability (data not shown), for this study, we selected a low-toxicity,high-efficiency fusion method using a Sendai virus envelope [20]. Aftersingle cell cloning, one of the clones, GMab-1 (IgG₃ subclass) wasestablished (FIG. 1B). As shown in FIG. 1C, GMab-1 reacted with3′-isoLM1 in a dose-dependent manner in ELISA. Furthermore, both D54cells and D54 xenograft cells, which express lacto-series gangliosideswere recognized by GMab-1 as demonstrated by flow cytometry (FIG.2A-2B). To determine the association and dissociation rate constants(k_(assoc) and k_(diss)) and to calculate the affinity constants(K_(A)), we next performed a kinetic analysis of the interaction ofGMab-1 with 3′-isoLM1 by surface plasmon resonance (BIAcore).Determination of the association and dissociation rates from thesensorgrams revealed a k_(assoc) of 5.07×10⁴ (mol/L-s)⁻¹ and a k_(diss)of 2.74×10⁻⁴ s⁻¹. The K_(A) at binding equilibrium, calculated asK_(A)=k_(assoc)/k_(diss), was 1.85×10⁸ (mol/L)⁻¹, indicating that GMab-1has high affinity against 3′-isoLM1.

Example 3 Specificity of GMab-1 Against Gangliosides

To determine the epitope of GMab-1, we performed ELISA using 15different gangliosides, including lacto-series (3′-isoLM1, 3′,6′-isoLD1,isoLA1, and Fuc-3′-isoLM1), neolacto-series (3′-LM1 and 3′,8′-LD1),globo-series (Gb3), asialo-series (GA1), a-series (GM2 and GM1), andb-series (GD3, GD2, GD1b, and GT1b) (A-3D. 3).

As previously reported [16,17], SL-50 reacted with 3′-isoLM1 and notwith 3′,6′-isoLD1 (FIG. 3C), whereas DMab-22 reacted with 3′,6′-isoLD1and not with 3′-isoLM1 (FIG. 3D). We have observed no cross-reactionwith the other gangliosides using SL-50 and DMab-22. DMab-14 reactedwith both 3′-isoLM1 and 3′,6′-isoLD1; however, there is slightreactivity with the other gangliosides (FIG. 3B). On the other hand, wedid observe activity that distinguished GMab-1 from SL-50, DMab-22, andDMab-14. GMab-1 recognized both 3′-isoLM1 and 3′,6′-isoLD1, and thereactivity with 3′,6′-isoLD1 was stronger than with 3′-isoLM1 (FIG. 3A).Although DMab-14 reacted with both 3′-isoLM1 and 3′,6′-isoLD1, thereactivity with 3′-isoLM1 was stronger than with 3′,6′-isoLD1 (FIG. 3B),which indicates that the epitope of GMab-1 is slightly different fromthat of DMab-14. Furthermore, there is no cross-reaction with the othergangliosides using GMab-1 (FIG. 3A). These results indicate that GMab-1defines the epitope NeuAca2-3Galβ1-3GlcNAc, the terminal sequence in3′-isoLM1 and 3′,6′-isoLD1.

Example 4 Immunohistochemical Analysis by GMab-1 Against GlioblastomaTissues

Since lacto-series gangliosides such as 3′-isoLM1 and 3′,6′-isoLD1 areknown to be expressed in glioblastoma tissues [18], immunohistochemistrywas performed using 33 glioblastoma frozen tissue specimens. GMab-1immunoreactivity was detected in 26 of 33 (78.8%) glioblastomas.Representative staining by GMab-1 in glioblastoma samples is shown inFIG. 4A-4D. Immunostaining by GMab-1 demonstrated predominantly bothcell-surface patterns (FIG. 4A) and cell-surface/cytoplasmic patterns(FIG. 4C) in glioblastoma cells. Proliferating endothelial cells werenegative for GMab-1 (FIG. 4A). The concentration-matched isotype controlof mouse IgG₃ did not stain any tumor cells (FIGS. 4B and 4D). Theseresults further suggest that GMab-1 specifically reacted withlacto-series gangliosides in glioblastoma tissues.

In conclusion, we immunized β3Gn-T5 knockout mice with 3′-isoLM1 and3′,6′-isoLD1. GMab-1 (SEQ ID NO:17 and 20), of the IgG₃ subclass,specifically recognized both 3′-isoLM1 and 3′,6′-isoLD1 and showed highbinding activity. GMab-1 was also reactive against glioblastomas inimmunohistochemical analyses, suggesting that GMab-1 should be useful inantibody-based therapy of glioblastomas.

Example 5 Additional Antibodies

We isolated an additional antibody produced by a hybridoma as describedin Example 2 above. Antibody GMab-2 (SEQ ID NO: 18 and 21) is of theIgG_(2b) subclass and specifically binds to both 3′-isoLM1 and3′,6′-isoLD1. Thus its binding specificity is similar to GMab-1. Bindingspecificities were tested by enzyme linked immunosorbent assays (ELISA).Control antibodies were the SL-50 IgM antibody which is specific for3′-isoLM1 and DMab-22, which is specific for 3′,6′-isoLD1.

GMab-2 did not bind to any of isoLA1, Fuc-3′-isoLM1, 3′-LM1, 3′,8′-LD1,LA1, Gb3, GM1, GD1b, GT1b, GA1, GD3, GM2, or GD2, defining its epitopicspecificity.

Example 6 Affinity Maturation

We constructed a recombinant single chain variable fragment (scFv) frompreviously isolated, hybridoma DMab14 (SEQ ID NO: 19 and 22). The IgMantibodies made by DMAB14 recognize both 3′iso-LM1 and 3′6′iso-LD1gangliosides, which are tumor associated antigens. Wikstand C J et al.,J. Neuropathol. Exp. Neurol. 1991, vol. 50:756-769. We fused the scFvwith Pseudomonas exotoxin A (PE38) at the carboxy terminus of the scFv.We investigated the binding affinity and specificity of the resultingrecombinant immunotoxin for gangliosides 3′iso-LM1 and 3′6′iso-LD1 onD54 MG cultured cells. These cells were previously shown to highlyexpress these gangliosides on their cell surfaces. The parental versionof DMab14-scFv showed a binding affinity of K_(d)=7.5×10-e7M to D54 MGbut did not demonstrate any cytotoxicity to cell line D54 MG. After fiverounds of affinity maturation by phage display, a new generation ofDMab14-scFv was generated, which was dubbed DMab14-86184-PEKDEL. (Forreview of display technologies including affinity maturation by phagedisplay, see Sergeeva et al., Adv. Drug Deliv. Rev. 2006, vol. 58:1622-1654.) DMab14-86184-PEKDEL contains a combination of mutations inthe VHCDR2 and in the VLCDR1. FIG. 5. The binding affinity ofDMab14-86184-PEKDEL to D54 MG cells was determined to beK_(d)=7.16×10-e8M. DMab14-86184-PEKDEL demonstrated cytotoxicity againstcell line D54 MG. In protein synthesis inhibition assays and cellproliferation assays DMab14-86184-PEKDEL displayed IC50=80 ng/ml andIC₅₀<1000 ng/ml, respectively. Moreover, when tested against the H336cell line, DMab14-86184-PEKDEL demonstrated a K_(d)=2.9×10-e7M and anIC50=10 ng/ml. In vivo studies are underway. Preliminary resultsindicate that DMab14-86184-PEKDEL has significant potential for treatingbrain tumors. Elimination of cells expressing 3′iso-LM1 and 3′6′-isoLD1should result in significant survival increases in brain tumor patients.

Materials and Methods:

Cloning of Variable Heavy (V_(H)) and Variable Light (V_(L)) Domains ofDMAb14.

We isolated total cellular mRNA from 10⁶ DMAb14-hybridoma cells by usingthe RNeasy Kit (Invitrogen, Carlsbad, Calif.). Primary V_(H) and V_(L)genes of the parental DMAb14 clone were amplified by RACE-PCR (RapidAmplification of cDNA Ends-PCR) using a SMART 5′-RACE cDNA amplificationkit (Clontech, Palo Alto, Calif.). The 3′ primers were mouse heavy-chain(HC) and light-chain (LC) constant region sequences of theimmunoglobulin (V_(H): 5′-GGC CAG TGG ATA GTC AGA TGG GGG TGT CGT TTTGGC-3′ (SEQ ID NO: 23) and V_(L): 5′-GGA TAC AGT TGG TGC AGC ATC-3′)(SEQID NO:24). The primary V_(H) and V_(L) cDNA genes obtained from 5′-RACEwere then used as templates to specifically amplify the V_(H) and V_(L)fragments respectively by using specific primers introduced atrestriction enzyme sites and a (Gly₄Ser)₅ linker sequence for scFvassembly and subsequent subcloning. The oligomers used for thesereactions were as follows: DMAb14-H-F (NcoI), 5′-GCC GCC ACCA TG Gag GTCCAA CTG CAG-3′(SEQ ID NO: 25); DMAb14-H-R (linker), 5′-AGA TCC GCC ACCACC GGA TCC CCC TCC GCC TGA GGA GAC GGT GAC-3′(SEQ ID NO: 26);DMAb14-L-F (linker), 5′-GGT GGT GGC GGA TCT GGA GGT GGC GGC AGC GGT AACATT GTG CTG-3′(SEQ ID NO: 27); and DMAb14-L-R (EcoRI), 5′-GCAGCC GAATTCAT TTT TAT TTC CAG CTT G-3′(SEQ ID NO: 28). We aligned and verified theoutcome-specific V_(H) and V_(L) sequences according to the Kabatalignment scheme. We constructed DMAb14-scFv using PCR splicingtechnology. In brief, we mixed Advantage 2 DNA polymerase (Clontech)with 50 ng each of V_(H) and V_(L) PCR fragments at a 1:1 ratio in thepresence of bovine serum albumin (BSA) in a 50-μL volume. The PCR mixwas cycled by using the following profile: 1 cycle at 96° C. for 5minutes; followed by 5 cycles each at 94° C. for 1 minute, 55° C. for 1minute, and 72° C. for 1 minute and 30 seconds; and a final cycle at 72°C. for 7 minutes. We paused the program at 4° C. for 2 minutes to addpremixed oligomers, namely, the forward and reverse primers that hadbeen used to generate the V_(H) and V_(L) fragments. The reaction wasthen continued as follows: 1 cycle at 96° C. for 5 minutes; followed by25 cycles each at 94° C. for 1 minute, 55° C. for 1 minute, and 72° C.for 1 minute and 30 seconds; and finally terminated at 72° C. for 7minutes. A 750-bp PCR product generated from this reaction was thenpurified by using Qiagen Quick Spin columns (Qiagen, Valencia, Calif.)and double digestion with NcoI and EcoRI and subcloned into a T7bacterial expression vector pET25b(+) (Novagen, Madison, Wis.). Weverified the parental DMAb14-scFv sequence by the dideoxychain-termination method.

Preparation of Recombinant Immunotoxins.

We generated the DMAb14-scFv IT by PCR using parental DMAb14-scFvplasmid as a template and primers introduced at the NdeI and HindIIIrestriction enzyme sites. After NdeI and HindIII digestion, we insertedthe scFv fragment into pRB199 bacterial expression vector that had beenengineered with the sequence for domains II and III of Pseudomonasexotoxin A (PE38KDEL) according to a previously described protocol (15).The parental DMAb14-scFv IT was expressed under control of the T7promoter in E. coli BL21 (λ DE3) (Stratagene, La Jolla, Calif.). Allrecombinant proteins remained in the inclusion bodies. We then reduced,refolded, and further purified the IT proteins as monomers (67 kDa) byusing ion-exchange and size-exclusion chromatography to greater than 90%purity.

Construction of DMAb14-scFv Phagemid.

We constructed the parental DMAb14-scFv phagemid by PCR amplificationfrom parental DMAb14-scFv-pRB199 PE38KDEL. We used the followingoligomers, which were introduced at the NcoI and NotI restriction enzymesites: DMAb14-scFv-F (NcoI), 5′-GCCGCCA CCATG GAG GTC CAA CTG CAG-3′(SEQID NO: 29), and DMAb14-scFv-R (NotI), 5′-ATG ATG TGC GGC CGC TTT TAT TTC CAG CTT G-3′(SEQ ID NO: 30). The PCR product was digested with NcoIand NotI and inserted into the phagemid vector pHEN2. We used theresulting parental phagemid DMAb14-scFv-pHEN2 as a template for furtherconstruction of mutant phage libraries.

Construction of V_(H) Mutant Library.

The V_(H)CDR2 of DMAb14 consisted of 18 amino acids. We designed DNAoligomers to generate 3 libraries, each randomizing 9 nucleotides (3consecutive amino acids). We used degenerate oligomers with the sequenceNNS for randomizing (N randomizing with all 4 nucleotides, and Sintroducing only C or G) (16). Parental DMAb14 phagemid was used as atemplate to introduce 3 amino acid randomizations in the CDR2 heavychain in 3 separate 2-step PCRs (FIG. 7A, 7B). We used the followingoligonucleotides: (a) DMAb14-VHCDR2a-R, 5′-GTT AGT ACG ACC GTT SNN SNNSNN AAT CTC TCC AAT CC-3′(SEQ ID NO: 31); (b) DMAb14-VHCDR2b-R, 5′-AATATA GTT AGT ACG SNN SNN SNN AGG ATT AAT CTC TCC-3′(SEQ ID NO: 32); (c)DMAb14-VHCDR2c-R, 5′-TAC AGT CAG TGT GGC SNN SNN SNN GAA CTT CTC-3′(SEQID NO: 33); (d) DMAb14-scFv-F (NcoI), 5′-GCCGCCA CCATG GAG GTC CAA CTGCAG-3′(SEQ ID NO: 34); and (e) DMAb14-scFv-R (NotI), 5′-ATG ATG TGC GGCCGC TTT TAT T TC CAG CTT G-3′(SEQ ID NO: 35). In the first PCR, we used50 pg of the phagemid DMAb14-scFv-pHEN2 as a template in 3 separatereactions involving 20 pmol of DNA oligomer DMAb14-scFv-F (NcoI), alongwith 20 pmol of DNA oligomer DMAb14-VHCDR2a-R, DMAb14-VHCDR2b-R, orDMAb14-VHCDR2c-R (FIG. 7A). The template and oligonucleotides were mixedwith Advantage 2 PCR Kit (Clontech) in a 50-μL volume and then cycledusing the following profile: 1 cycle at 95° C. for 5 minutes, followedby 30 cycles each at 94° C. for 1 minute, 55° C. for 1 minute, and 72°C. for 1 minute. This reaction generated 280-bp products that containedmutations. The products were purified by using Qiagen Quick Spin columns(Qiagen, Inc.) and then quantified by visualization on a 1% agarose gel.We used the purified products generated in the first PCR as primers in asecond PCR. In the second reaction, we used approximately 2 pmol each ofthe 3 products containing mutations obtained from the first reaction andthe NcoI restriction enzyme site, along with 20 pmol of the DNA oligomerDMAb14-scFv-R (NotI) and 50 pg of the parental phagemidDMAb14-scFv-pHEN2 as a template to generate the whole length of thescFv-V_(H) mutation pool (FIG. 7B). The PCRs were set and cycled byusing the profile described above. Each reaction generated 750-bp insertlibraries. The PCR product was digested with NcoI and NotI and purifiedby using Qiaquick columns (Qiagen, Inc.). The purified PCR product (80ng) was ligated with 150 ng of the phage display vector pHEN2(predigested with NcoI and NotI) and desalted. We used 40 ng of ligatedproducts to transform E. coli TG1 (Stratagene, La Jolla, Calif.) byelectroporation. We performed 3 transformations to create a librarycontaining 6×104 clones. We then rescued the phage libraries from thetransformed bacteria. Cells from each transformation were grown in 10 mLof 2×YT (Bacto Yeast Extract and Bacto Tryptone) containing 2% glucoseat 37° C. with shaking at 250 rpm. After 1 hour, we added ampicillin(100 μg/mL, final concentration) and 1×10¹¹ plaque-forming units(pfu)/mL of VCSM13 Interference-Resistant Helper Phage (Stratagene,Agilent Technologies, Palo Alto, Calif.). The cultures were grown for 1hour, pelleted, resuspended in 10 mL of 2×YT supplemented withampicillin (100 μg/mL) and kanamycin (50 μg/mL), and grown for 16 hoursat 30° C. with shaking at 250 rpm. The bacteria were pelleted bycentrifugation in a Sorvall SS34 rotor at 8000 rpm for 20 minutes. Thephage-containing supernatants were filtered through a 0.45-μm syringefilter unit. The phages were precipitated by adding 2 mL of PEG/NaCl(20% PEG8000 in 2.5M NaCl [w/v]) and incubated on ice for 30 minutes.The precipitated phages were pelleted by centrifugation in a SorvallSS34 rotor at 10,000 rpm for 20 minutes and resuspended in 1 mL of NTE[100 mM NaCl, 10 mM Tris (pH 7.5), and 1 mM EDTA]. The rescued phagelibraries were titered and stored at 4° C.

Screening of V_(H) Mutant Library by Phage ELISA.

We cultured and plated a diluted V_(H)CDR2 mutant phage library.Recombinant phage-containing supernatants were rescued from individualclones and screened by antigen-based phage ELISA. We picked singlecolonies from the diluted library plates and inoculated them into 1 mLof 2×YT medium supplemented with 2% glucose and 100 μg/mL of ampicillin.The colonies were cultured overnight at 37° C. with shaking. To onecultured colony preparation, we added 450 μL of 2×YT supplemented with2% glucose and 100 μg/mL of ampicillin contained 2.5×10⁸ pfu of VCSM13Interference-Resistant Helper Phage (Stratagene) in 1.2-mL Cluster Tubes(Corning Incorporated, Corning, N.Y.). To another preparation, we added50 μL of the overnight culture and then incubated the colony at 37° C.for 2 hours with shaking at 250 rpm. The cells were pelleted andresuspended in 500 μL 2×YT containing 100 μg/mL ampicillin and 50 μg/mLkanamycin without glucose and then grown for 16 hours at 30° C. Thecells were again pelleted by centrifugation at 2800 rpm at 4° C., andthe phage-containing supernatants were saved.

Purified gangliosides 3′-isoLM1 and 3′6′-isoLD1 conjugated with BSA(purified from the D54 MG xenograft, Sahlgrenska University Hospital,Gothenburg, Sweden) in PBS were immobilized overnight in 96-well plates(Nunc, Thermo Fisher Scientific, NY) containing 50 ng/well at roomtemperature. We blocked the plates with 1% BSA/PBS at 37° C. for 30minutes and then washed them 3 times with 0.05% Tween 20 in PBS by usinga Bio-Rad Auto-Washer (Bio-Rad 1575 Immuno Wash Microplate Washer). Wethen added 100 μL/well of phage-containing supernatant for a 1-hourincubation at 37° C. and washed the plates 3 times as above. For phageELISA, we added 100 μL/well of anti-M13 antibody-horseradish peroxidase(HRP) conjugate (Pharmacia Biotech, Sweden) in blocking buffer, dilutedaccording to the manufacturer's instructions, to detect phage binders.For monoclonal IgM antibody, we added 100 μL/well of anti-mouse IgM HRPconjugate (μ-Chain Specific; Sigma, St. Louis, Mo.) in blocking buffer(1:10,000 dilution). After 30 minutes of incubation at 37° C., we washedthe plates and used 100 μL/well of 3,3′,5,5′-tetramethyl benzidine(1-Step Ultra TMB-ELISA, Pierce, Rockford, Ill.) solution as a substratefor detection. After a blue color developed, we added 50 μL TMB StopSolution (Pierce) to stop further color development. We measuredabsorption at 450 nm. Clones reacting to gangliosides were referred toas positives.

Construction of V_(L) Mutant Library.

The V_(L)CDR1 of DMAb14 consists of 15 amino acids. We used parentalDMAb14 phagemid DNA as a template in 4 separate 2-step PCRs thatintroduced randomizations in the hot spot located in the light-chainCDR1 (FIG. 7A, 7C). We used the following oligonucleotides: (a)DMAb14-VLCDR1a-F, 5′-ACC ATA TCC TGC NNS NNS NNS GAA AGT GTT GAG AGT TATGGC-3′(SEQ ID NO: 36); (b) DMAb14-VLCDR1b-F, 5′-ACC ATA TCC TGC AGA NNSNNS NNS AGT GTT GAG AGT TAT GGC-3′(SEQ ID NO: 37); (c) DMAb14-VLCDR1c-F,5′-ACC ATA TCC TGC AGA GCC NNS NNS NNS GTT GAG AGT TAT GGC-3′(SEQ ID NO:38; (d) DMAb14-VLCDR1d-F, 5′-GCC AGT GAAAGT GTT NNS NNS NNS GGC AAT AATTTT ATG CAC-3′(SEQ ID NO: 39); (e) DMAb14-scFv-F (NcoI), 5′-GCCGCCACCATG GAG GTC CAA CTG CAG-3′(SEQ ID NO: 40; and (f) DMAb14-scFv-R(NotI), 5′-ATG ATG TGC GGC CGC TTT TAT T TC CAG CTT G-3′(SEQ ID NO: 41).In the first reaction, we mixed 50 pg of the DMAb14-scFv phagemid DNAwith 20 pmol of each pair of DNA oligomers in a 50-μL volume as follows:(a)+(f), (b)+(f), (c)+(f), and (d)+(f). The mixture was cycled by usingthe same profile used to generate the heavy-chain CDR2 mutant library.The reactions generated 260-bp products exhibiting randomization of thehot spot in the light-chain CDR1. After purification and quantification,2 pmol of the 260-bp fragments generated in the first PCR, was subjectedto a second PCR, along with 20 pmol of DNA oligomer (e) and 50 pg ofparental DMAb14-scFv phagemid DNA as a template (FIG. 7C). We carriedout the PCR (volume, 50 μL) by using an Advantage 2 PCR Kit (Clontech)and following the above-mentioned profile. The reactions generated a750-bp library, which showed randomization of the hot spot in V_(L)CDR1.The PCR products were then digested with the restriction enzymes NcoIand NotI, purified, and ligated with the pHEN2 phage vector as describedfor the heavy-chain CDR2 mutant libraries. We desalted the ligation andused one-tenth (40 ng) of the reaction to transform E. coli TG1. Werescued the V_(L)CDR1 mutant phage library containing 5×10⁴ clones asdescribed in the heavy-chain CDR2 construction.

Selection of V_(L)CDR1 Library by Phage ELISA.

We cultured and plated a diluted V_(L)CDR1 phage library. The technicalprocedures used were the same as those described in the V_(H)CDR2libraries selection.

Construction of V_(H)CDR2 and V_(L)CDR1 Combination Libraries.

We used the heavy-chain CDR2 mutant as a template to generate the V_(H)fragment. For this purpose, we used the primers DMAb14-H-F (NdeI), 5′-GGCG CAT ATG CAT GTC CAA CTG CAG-3′ (SEQ ID NO: 42) and DMAb14-H-R(linker), and 5′-AGA TCC GCC ACC ACC GGA TCC CCC TCC GCC TGA GGA GAC GGTGAC-3′ (SEQ ID NO: 43) in a 50-μL volume and an Advantage 2 PCR Kit(Clontech) and obtained a 420-bp product. We used the light-chain CDR1mutant as a template to generate the V_(L) fragment. We used the primersDMAb14-L-F (linker), 5′-GGT GGT GGC GGA TCT GGA GGT GGC GGC AGC GGT AACATT GTG CTG-3′(SEQ ID NO: 44) and DMAb14-L-R (HindIII), and 5′-C AAG CTGGAA ATA AAA A AA GCT TGG CAG C-3′(SEQ ID NO: 45) in a 50-volume and anAdvantage 2 PCR Kit (Clontech) to generate a 335-bp PCR product. mutantV_(H) and mutant V_(L) DNA fragments were overlapped by a 15-amino acidlinker sequence and connected by using the splicing PCR techniquedescribed above, except that the oligomers used in this reaction wereDMAb14-H-F (NdeI) and DMAb14-L-R (HindIII), which are listed above inthis section. This reaction generated 750-bp products that containedmutations in both the V_(H) and V_(L) regions. The products werepurified by using Qiagen Quick Spin columns (Qiagen, Inc.), digestedwith NdeI and HindIII, and cloned into a T7 expression vector pRB199 inwhich scFv was fused to a truncated version of Pseudomonas exotoxin A(PE38KDEL). Their sequence was then verified.

Analysis of Selected Clone by DNA Sequencing.

Sequencing was performed by the Duke University DNA Sequencing Facilityon an Applied Biosystems Dye Terminator Cycle Sequencing system (LifeTechnologies, Corp., Carlsbad, Calif.) by using AmpliTaq DNA Polymeraseand ABI 3730 PRISM DNA Sequencing instruments. Sequencing analysis wasperformed using the NCBI nucleotide BLAST program.

Cell-Binding Assay and Constant K_(d) by FACS.

We used a modification of the method reported previously (17) to detectscFv immunotoxin binding to ganglioside-expressing cells. To preventinternalization of the immunotoxin during the assays, we kept allreagents and the buffer on ice. In brief, cells to be tested for scFvimmunotoxin binding were harvested by trypsinization, rinsed with 1% FBSin PBS, and portioned to assay tubes (approximately 10⁶ cells persample). The scFv immunotoxins were serial-diluted in 100 μL of 1%FBS-PBS, with an initial amount of 25.6 μg. The cells were incubatedwith 100 μL of various dilutions of the purified scFv immunotoxin oranti-Tac(Fv)-PE38-KDEL, a negative control that does not bind togangliosides, for 1 hour on ice. We then washed the cells twice in 1%FBS-PBS and incubated them with rabbit anti-Pseudomonas exotoxin Aantibody (Sigma) for 30 minutes on ice. After the cells were washedtwice, we reacted them with goat anti-rabbit IgG-FITC conjugate (Sigma)for 30 minutes on ice. The stained cells were again washed twice andthen subjected to flow cytometry. We used an identical protocol forbinding of the scFv immunotoxin in this study. All reagents weredetermined to be at saturation point, and binding reached equilibriumfor the described conditions in all antibody-binding experiments. Wedetermined equilibrium constants by using the Graph Pad Prism Software(version 4.0) for 1-site binding and nonlinear regression analysis.

Determination of Affinity Constants (K_(d)) by Scatchard Analysis.

We performed affinity measurements by using Iodogen-labeled scFvimmunotoxin binding to cells positive and negative for gangliosideexpression. We seeded the cells in a 24-well plate, cultured them untilconfluence, fixed them in 0.25% gluteraldehyde for 5 minutes at roomtemperature, washed them 3 times with incubation buffer (115 mM PO₄[KH₂PO₄+K₂HPO₄] buffer with 0.05% BSA and 0.05% gelatin), and froze themat −80° C. until use. [¹²⁵I]-labeled sample scFv and negative controlscFv immunotoxin were serial diluted starting from 8 μg/mL to 4 ng/mL,followed by the addition of various dilutions of I¹²⁵-scFv immunotoxinsto corresponding triplicate wells and incubation of the cell platesovernight at 4° C. The next day, we removed the free radioactiveantibody and washed the cell plates 4 times with the incubation buffer.We then added 500 μL/well of 2N sodium hydroxide and incubated the cellplates overnight at 37° C. The cells were completely suspended andtransferred (including cell debris) into 2-mL screw-capped tubes, whichwere placed in a gamma counter in order, and the cells were counted. Thedata obtained were analyzed by normalization based on standard curve andequilibrium constants, and a Scatchard plot was determined with Prismsoftware (version 4.0) for nonlinear regression analysis.

Cytotoxicity Assays.

We measured cytoxicity using both protein synthesis inhibition and celldeath assays. In both assays, we plated cells in 96-well plates (Nunc)at a concentration of 1×10⁴ cells/200 μL/well. We serially dilutedimmunotoxins in PBS supplemented with 0.2% BSA starting from 10 ng/4 andadded 20 μL of each dilution to the corresponding wells, resulting in amaximum immunotoxin concentration of 1000 ng/mL. We incubated the platesfor 20 hours at 37° C.

We measured protein synthesis on the basis of [³H] leucineincorporation. We pulsed the cells with 1 μCi/well of [³H] leucine in 20μL of PBS supplemented with 0.2% BSA for 2.5 h at 37° C. We capturedradiolabeled cells on a FilterMate micro-harvester (PerkinElmer Life andAnalytical Sciences, Boston, Mass.) and counted them with a Betaplatescintillation counter (Amersham Biosciences, GE Healthcare Bio-SciencesCorp., Piscataway, N.J.). Triplicate sample values were averaged, andthe inhibition of protein synthesis was determined by calculating thepercentage of incorporation compared with control wells without addedtoxin. The activity of the molecule was defined as the IC₅₀.

We assessed cell death by WST-8 conversion using Cell Counting Kit-8according to recommendations from the supplier (Dojindo MolecularTechnologies, Gaithersburg, Md.). We seeded the cells onto a 96-wellplate (Costar, Corning Incorporated) at a concentration of 2×10⁴cells/100 μL/well. We serially diluted the immunotoxins in PBSsupplemented with 0.2% BSA starting from 10 ng/4 and added 10 μL of eachdilution to the corresponding wells. The cells were then incubated at37° C. for 20 h. We added 10 μL of WST-8 (5 mM WST-8, 0.2 mM1-methoxy-5-methylphenazinium methylsulfate, and 150 mM NaCl) to eachwell, and continued the incubation at 37° C. We measured the absorbanceof the sample at 450 nm every 20 minutes until the highest value for thenegative control was obtained. Cytotoxicity was expressed as 50%inhibition of cell viability. We performed all experiments intriplicate. Statistical analyses were performed with Prism software(version 4.0) for Windows (GraphPad software, San Diego Calif.). Eachimmunotoxin was assayed at least twice, and critical immunotoxins wereassayed more frequently.

DMAb14-scFv-Mut Epitope Mapping.

DMAb14-scFv immunotoxins that specifically bind to gangliosides3′-isoLM1 and 3′6′-isoLD1 were assayed by ELISA. We coated each purifiedganglioside (500 pM) in PBS on a 96-well plate (Nunc), which was blockedwith 1% BSA/PBS at 37° C. for 1 h. We applied 500 ng primary antibodiesin triplicate and incubated the plate at 37° C. for 30 minutes. We usedD2C7 immunotoxin as a scFv structural negative control, SL50 IgM andDMAb22 IgM as positive controls for 3′-isoLM1 and 3′6′-isoLD1,respectively, and MOPC as an IgM negative control. We washed the plate 3times with 0.05% BSA and 0.05% Tween in PBS. After adding rabbit anti-PE(Sigma; dilution 1:30,000) to the scFv immunotoxins for 30 minutes andwashing the plate, we applied anti-rabbit IgG-HRP (Sigma, dilution1:1,500) and anti-mouse IgM-HRP (Sigma, dilution 1:1,500) to the scFvimmunotoxins and IgMs, respectively. The plate was incubated at 37° C.for 30 minutes and washed 3 times with a washing buffer. We then added100 μL of 1-Step Ultra-TMB ELISA (Thermal Scientific, Odessa, Tex.)substrate, and the reaction color turned blue within 3-5 minutes; weimmediately applied stop solution to terminate the reaction and changethe color to yellow. The ELISA plate was read at an absorbance of 450nm.

Internalization Assay.

We directly labeled DMAb14-scFv immunotoxins with Alexa-488 andincubated 50 nM Alexa-488-labeled DMAb14-scFv immunotoxin with 3×10⁵H336 cells or Xeno-2224 cells on ice for 1 hour. After washing the cellstwice with ice-cold PBS, we resuspended the cells in zinc option medium.Leaving one portion of the reaction mix for measurement of backgroundinternalization and one portion for total binding measurement on ice, weincubated the rest of the reaction mix at 37° C. for 15 minutes, 30minutes, and 1 h, 2, 4, and 8 hours. We washed the cells twice withice-cold PBS and in a cold centrifuge. Except for the total bindingportion, we resuspended all cells in 500 nM quenching anti-Alexa-488antibody (Invitrogen) diluted in ice-cold PBS. All tubes were incubatedfor 1 hour on ice. We washed the cells twice in ice-cold PBS andresuspended them in 4% paraformaldehyde; we analyzed the reactionsamples in a flow cytometer (FACSCalibur, BD Biosciences, FranklinLakes, N.J.). We calculated internalization as the fluorescence ofquenched cells (intracellular compartments only) divided by that ofunquenched cells (both cell surface and intracellular compartments;total binding) after normalization against the background fluorescence.The cells incubated with Alexa-488-labeled immunotoxins on ice were usedas a control while estimating the antibody's quenching efficiency oneach Alexa-labeled immunotoxin. Since internalization should not occurat 0° C., the fluorescence of these cells measured after quenching withanti-Alexa-488 was considered to represent the unquenchable surfacefluorescence (background). This amount was corrected when the percentageinternalized immunotoxin was calculated.

Cell Culture.

We used the human malignant glioma-derived cell lines D54 MG and

H336, which were established and maintained in our laboratory, asganglioside-expressing cell lines. We used the human embryonic kidneycell line H293 as a negative control. We cultured all cell lines inImproved MEM Zinc Option (Richter's zinc option; Invitrogen)supplemented with 10% FBS (Invitrogen) and passed at confluence with0.25% trypsin-EDTA (Invitrogen).

Disaggregation of Xenograft Tumor Samples.

Xenograft tissues from malignant gliomas (H2224 and BT56) obtained understerile conditions from animals housed in the Duke Cancer CenterIsolation Facility were prepared for cell culture in a laminar flow hoodby using a sterile technique. Tumor material was finely cut withscissors and added to a trypsinizing flask containing approximately 10mL of 100 μg Liberase (Roche, Indianapolis, Ind.). This mixture wasstirred at 37° C. for 10 minutes, and cell-rich supernatant wasobtained. We filtered the dissociated cells through a 100-μm cellstrainer (BD Falcon, BD Biosciences), washed them with complete medium,and pelleted them at 1000 rpm for 5 minutes. We further treated the cellsuspension with Ficoll-Hypaque to remove any red blood cells and thenwashed it once in complete medium. We cultured and passaged the cellsuntil sufficient numbers were obtained and harvested them with 0.25%trypsin-EDTA.

Results:

Construction of the V_(H)CDR2 and V_(L)CDR1 Libraries.

We improved the affinity of DMAb14 (scFv) through random mutagenesisbased on hot spots within either the V_(H)CDR2 region or the V_(L)CDR1region, because these portions of the antibody might have significantcontact with the antigens. The amino acid sequences of V_(H)CDR2 andV_(L)CDR1 are shown in Table 5. The libraries introduced randomizationsin the hot spot motif residues S75, N76, G77, K86, S87, and K88 forV_(H)CDR2 and R25, A26, S27, S32, and Y33 for V_(L)CDR1 in separateexperiment sets as described in “Materials and Methods.” In the step ofcloning, 3 V_(H) libraries were mixed and yielded 6×10⁴ clones, and 4V_(L) libraries yielded 5×10⁴ clones. We sequenced 20 clones from theV_(H) library and 14 from the V_(L) library to verify that theconstruction of the library was appropriate. Sequencing showed that eachclone had different amino acid combinations in the region targeted formutations. The size of the libraries ensured that most of the DNAsequences were represented (data not shown).

TABLE 5Nucleotide and amino acid sequence of heavy-chain CDR2 and light-chain CDR1 withhot spots of DMAb14 are shown* DMAb14 GAG ATT AAT CCT AGC AAC GGT GGTGGT AAC TAT AAT GAG AAG TTC AAG AGC AAG V_(H)CDR2 E I N P S N G R T N YN E K F K S K Position 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 8687 88 DMAb14 AG A GC C AGT GAA AGT GTT GAG AGT T AT GGC AAT AAT TTT ATGCAC V_(L)CDR1 R A S E S V E S Y G N N F M H Position 25 26 27 28 29 3031 32 33 34 35 36 37 38 39 *Hot-spot positions are indicated withunderline and boldface. Sequences shown on lines 1, 2, 4, and 5 of theTable are SEQ ID NOS: 46-49, respectively.

Selection of V_(H)CDR2 and V_(L)CDR1 Libraries and Analysis of SelectedClones.

We rescued recombinant phages from 500 individual phage clones from eachlibrary, and we randomly selected and tested these phages for reactivityagainst purified gangliosides by phage ELISA. We found that 10 clonesfrom the V_(H) library and 8 clones from the V_(L) library showed highsignals on ELISA. We repeatedly tested the selected clones with phageELISA and sequenced them, and we finally found two clones from the V_(H)library and three from the V_(L) library that constantly exhibitedstrong signals during reaction with gangliosides and containedfull-length scFv without stop codons (Table 6). V_(H)CDR2 mutant clones86-MDS and 126-LPP retained the same residues as the parental V_(H) atpositions 86-88, but these two clones exhibited mutations at S75M, N76D,G77S and S75L, N76P, G77P, respectively. The V_(L)CDR1 mutant clone23-RA showed mutations at S27R and S29A, whereas the mutant clones104-PWR and 184-LYG showed PWR and LYG respectively at positions 25-27in place of RAS in the parental clone (Table 6).

TABLE 6Positive clones were selected from the V_(H)CDR2 and V_(L)CDR1 libraries*IC₅₀ K_(d) Position:  71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 8788 (ng/ml) (μM) DMAb14-V_(H)CDR2 Original Amino Acid E I N P S N G R T NY N E K F K S K N/A N/A DMAb 14-V_(H)CDR2 Mutant^(#) No. 86 E I N P M DS R T N Y N E K F K S K N/A     0.9 No. 126 E I N P L P P R T N Y N E KF K S K N/A 280 Position: 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39DMAb14-V_(L)CDR1^(#) Original Amino Acid R A S E S V E S Y G N N F M HN/A N/A DMAb14-V_(L)CDR1Mutant No. 23 R A R E A V E S Y G N N F M H N/A    0.8 No. 104 P W R E S V E S Y G N N F M H N/A     4.7 No. 184 L Y GE S V E S Y G N N F M H N/A     0.2 *Hot-spot positions are indicatedwith underline and boldface. ^(#)When VHCDR2 had mutations, VLCDR1 keptparental residues. When VLCDR1 had mutations, VHCDR2 kept parentalresidues. Lines of sequence 1 2, 3,4, 5, 6, and 7 of the table show SEQID NO: 50-56, respectively

Binding Properties and Cytotoxicity of V_(H)CDR2 and V_(L)CDR1 Mutants.

We prepared V_(H)CDR2 and V_(L)CDR1 mutant immunotoxin clones andmeasured their cytotoxic activities and binding affinities usingpurified recombinant immunotoxin proteins on D54 glioblastoma cells. TheV_(H)CDR2 mutants did not show improved binding affinity. Mutant clone86-MDS, with mutations at positions V_(H) 75-77, had an affinity ofK_(d)=0.9 μM, and the affinity of clone 126-LPP (K_(d)=280 μM) wascomparable to that of the parental clone (K_(d)=0.45 μM) (Table 6). Ofthe three V_(L)CDR1 mutants, clone 184-LYG showed better bindingaffinity (K_(d)=0.2 μM) than both the parental and the V_(H)CDR2 mutantclones. The other two V_(L)CDR1 mutant clones, 23-RA (K_(d)=0.8 μM) and104-PWR (K_(d)=4.7 μM), did not display improved affinity as comparedwith the parental and V_(H)CDR2 mutant clones (Table 6). None ofV_(H)CDR2 and V_(L)CDR1 mutant immunotoxins showed any significantcytotoxic activity in the protein-synthesis inhibition assay (data notshown).

Construction of Combination V_(H)CDR2 and V_(L)CDR1 Mutant.

Our goal was to obtain a scFv with increased affinity for gangliosides,which when converted to an immunotoxin would improve cytotoxic activitytoward cells expressing gangliosides. Neither V_(H)CDR2 nor V_(L)CDR1affinity-matured mutants showed improved binding affinity when comparedwith that of the parental clone or any significant cytotoxic activity.We therefore combined the V_(H) and V_(L) mutants to form a scFv withmutations in both V_(H)CDR2 and V_(L)CDR1. We constructed sixrecombinant clones as described in “Materials and Methods” and preparedthe immunotoxins as the following scFvs: DMAb14-8623-PEKDEL,DMAb14-86104-PEKDEL, DMAb14-86184-PEKDEL, DMAb14-12623-PEKDEL,DMAb14-126104-PEKDEL, and DMAb14-126184-PEKDEL (Table 7). We purifiedeach immunotoxin to over 90% homogeneity and eluted it as a monomer byusing TSK gel filtration chromatography (Sigma-Aldrich Co., St. Louis,Mo.) (results not shown). We used the purified immunotoxins incytotoxicity assays and binding property measurement by flow cytometry.

TABLE 7 The combination mutant clones have mutations in both VHCDR2and VLCDR1* V_(H)CDR2 DMAb14-8623 E I N P M D S R T N Y N E K F K S KDMAb14-86104 E I N P M D S R T N Y N E K F K S K DMAb14-86184 E I N P MD S R T N Y N E K F K S K DMAb14-12623 E I N P L P P R T N Y N E K F K SK DMAb14-126104 E I N P L P P R T N Y N E K F K S K DMAb14-126184 E I NP L P P R T N Y N E K F K S K V_(L)CDR1 DMAb14-8623 R A R E A V E S Y GN N F M H DMAb14-86104 P W R E S V E S Y G N N F M H DMAb14-86184 L Y GE S V E S Y G N N F M H DMAb14-12623 R A R E A V E S Y G N N F M HDMAb14-126104 P W R E S V E S Y G N N F M H DMAb14-126184 L Y G E S V ES Y G N N F M H *Hot-spot positions are indicated with underline andboldface. Lines of VHCDR2 sequence are SEQ ID NO: 57-62, respectively,and lines of VLCDR1 are SEQ ID NO: 63-68, respectively.

Binding Affinity of Combined Mutants Toward Cell Lines.

To determine whether the increased activity of the combined mutantimmunotoxins was attributable to increased binding affinities, wemeasured affinity by using established flow cytometry as described in“Materials and Methods.” The binding affinities of the six combinedaffinity-matured DMAb14 immunotoxins toward D54 MG cells are summarizedin Table 8; these immunotoxins were found to be capable of binding toganglioside-expressing cells on flow cytometric analysis. Two of thecombined mutant immunotoxins, DMAb14-86184 and DMAb14-12623, showedincreased binding affinity toward D54 MG cells, about 7 to 35 timeshigher than that of the V_(H)CDR2 or V_(L)CDR1 positive clones alone(Table 6) and about 10 to 15 times higher than that of the parentalDMAb14 immunotoxin (profile data not shown). The mutant with the highestaffinity was DMAb14-86184-MDSLYG, with a K_(d) of 26.5 nM. DMAb14-86-MDSshowed a K_(d) of 900 nM, DMAb14-184-LYG a K_(d) of 200 nM, and parentalDMAb14 a K_(d) of 450 nM. The mutant DMAb14-12623 showed a K_(d) of 28.8nM and had a similar pattern to that of DMAb14-86184-MDSLYG. The rest ofthe affinity-matured mutants did not show significantly improved bindingaffinities.

TABLE 8 Binding affinities and cytotoxic activities of combinationmutant immunotoxins on the D54 MG cell line differ* Kd (nM) IC₅₀ (ng/mL)DMAb14-Parental 450 N/A DMAb14-8623 549.4 N/A DMAb14-86104 352.5 N/ADMAb14-86184 26.5 80 DMAb14-12623 28.8 N/A DMAb14-126104 1420 N/ADMAb14-126184 127.1 N/A *Boldface denotes affinity-matured mutants withsignificantly improved binding affinity.

Cytotoxic Activity of Combined Mutant Immunotoxins on Cell Lines.

We next investigated the cytotoxic activity of the combined mutantimmunotoxins on ganglioside-expressing D54 MG cells (FIG. 8). Wepurified each immunotoxin to over 90% homogeneity and eluted it as amonomer using TSK gel filtration chromatography (results not shown). Asshown in FIG. 8, only one of the six combined affinity-matured mutantimmunotoxins, namely, DMAb14-86184-PEKDEL, had cytotoxic activity on D54MG cells, with an IC₅₀ of 80 ng/mL. The other combined mutantimmunotoxins did not show significant cytotoxic activities on D54 MGcells (data not shown). Thus, we selected clone DMAb14-86184-PEKDEL,renaming it DMAb14-scFv-Mut-PEKDEL, for further studies.

DMAb14-scFv-Mut IT Epitope Mapping.

DMAb14-scFv ITs specifically bind to gangliosides 3′-isoLM1 and3′6′-isoLD1, as shown in FIG. 9. Both DMAb14-scFv parental andDMAb14-scFv-Mut ITs showed significantly higher signals upon reactionwith gangliosides 3′-isoLM1 and 3′6′-isoLD1 than those shown by thestructural negative control D2C7-IT. We used different reaction systemsfor scFv-IT and positive IgMs; nonetheless, significantly higher signalswere obtained for SL50 IgM (antibody for 3′-isoLM1) and DMAb22 IgM(antibody for 3′6′-isoLD1) than for negative control MOPC IgM on3′-isoLM1 and 3′6′-isoLD1, respectively, indicating that thegangliosides were properly coated.

Cytotoxic Activity of DMAb14-scFv-Mut Immunotoxin on Cell Lines andXenografts.

We further tested the cytotoxic activity of DMAb14-scFv-Mut-PEKDEL ondifferent cell lines and xenografts known to express cell-surfacegangliosides. We evaluated cytotoxicity (IC₅₀) on the basis of both theprotein synthesis inhibition assay ([³H] leucine incorporation) and thecell death assay (WST-8) (FIG. 10). The parental DMAb14-scFv-PEKDEL didnot display any cytotoxic activity on any ganglioside-expressing cells.In contrast, the affinity-matured DMAb14-Mut-IT exhibited an IC₅₀ of 2ng/mL (29.9 pM) toward H336 cells, a malignant glioblastoma cell line(FIG. 10A, 10B), and an IC₅₀ of 0.5 ng/mL (7.5 pM) toward xenograftcells 2224, also derived from a malignant glioblastoma (tumor block fromDuke Hospital) (FIGS. 10C, 10D). DMAb14-Mut-IT showed the highestcytotoxic activity toward xenograft cells 2224, 4-fold greater than itsactivity on H336 cells. The mutant immunotoxin was not cytotoxic to theganglioside-negative cell line D293 (a human embryonic kidney 293 cellline established by Duke University; data not shown), indicating thatthe cytotoxic effect of this immunotoxin is selective toantigen-positive cells.

Binding of DMAb14-scFv-Mut Immunotoxin to H336 Cells.

In order to accurately determine the dissociation constant value, weperformed a cell-binding assay using isotope-direct-labeled immunotoxinreacting with ganglioside-expressing H336 cells. DMAb14-Mut immunotoxinwas labeled with iodine 125, and its binding to live H336 cells at 4° C.was assayed as described in “Materials and Methods.” The data wereplotted for Scatchard analysis (FIG. 11); the slope of the plot thusobtained was 0.38 nM⁻¹, giving a K_(d) of 2.6 nM, and its regressioncoefficient was 0.982. This K_(d) value is 10 times lower than thatmeasured by flow cytometry of the same H336 cells.

Internalization of DMAb14-scFv-Mut Immunotoxin in H336 Cells.

We quenched surface fluorescence by using anti-Alexa-488 antibody inorder to determine the internalized fraction. Internalization wasmeasured as a percentage of the total amount bound to gangliosidebinders by comparing the Alexa-488 fluorescence of the quenched cells(intracellular compartment) to that of the unquenched cells (bothintracellular and cell-surface compartments) by using flow cytometricanalysis. Unquenchable surface fluorescence (background) was estimatedin parallel tubes on ice (no internalization at 0° C.). The amount ofunquenchable fluorescence of Alexa-488-labeled DMAb14-scFv-Mut andDMAb14-scFv parental immunotoxins was about 24% and 8.5%, respectively,when incubated with H336 cells and 13% and 1.2%, respectively, whenincubated with xenograft 2224 cells (FIG. 12). We corrected eachindividual experiment for this “background fraction” before calculatingthe internalization percentage.

As the duration of incubation at 37° C. increased, the internalizationpercentage of DMAb14-scFv-Mut-IT in H336 cells increased gradually atfirst (0.82% at 15 minutes, 14% at 30 minutes, 16% at 1 hour, 30% at 2hours) and then dramatically increased to 98% at 4 hours, eventuallyreaching 100% at 8 hours. In contrast, the internalization percentage ofDMAb14-scFv parental immunotoxin fluctuated between 6% and 12% and wasnot significant throughout the incubation period (FIG. 12A).DMAb14-scFv-Mut-IT internalization into xenograft 2224 cells beganincreasing after 30 minutes and reached 43% at 1 hour, 80% at 2 hours,94% at 4 hours, and 100% at 8 hours. The DMAb14-scFv parentalimmunotoxin did not show significant internalization in the xenograft2224 cells (FIG. 12B).

Discussion:

Our goal was to develop an anti-ganglioside scFv immunotoxin with highcytotoxic activity and tumor specificity. The approach used to increasethe affinity of the parental MAb, DMAb14-scFv, was to mutagenize hotspot residues in V_(H)CDR2 and V_(L)CDR1. Hot spots are regions of DNAthat are frequently mutated during the in vivo affinity maturation ofantibodies. In contrast with other strategies for increasing antibodyaffinity, our approach of targeting hot spots allowed us to make a smalllibrary (4×10⁴ clones) that covered all possible mutations. We were ableto identify several different mutations that conferred increasedaffinity, and when the mutations in V_(H)CDR2 and V_(L)CDR1 werecombined in a single clone, the antibody's affinity was even higher.

While the analysis of mutant clones selected by phage ELISA screeningrevealed the presence of a variety of different sequences, there was apattern to the amino acid substitutions (Table 6). In the mutant clones,three amino acids of V_(H)CDR2 (75-77) and three of V_(L)CDR1 (25-27)were consistently different from the parental sequence, which indicatesthat these residues are required for interacting with the antigens. Whenthe three mutations in V_(H)CDR2 and the three mutations in V_(L)CDR1were combined in one scFv construct (DMAb14-scFv-86184-PEKDEL, hereafterreferred to as DMAb14-scFv-Mut-IT), the clone showed improved bindingaffinity (Tables 7 and 8) and dramatic cytotoxic activity (FIG. 8 andFIG. 10). It is possible that the V_(H) and V_(L) loop may require allthese residues together for proper conformational flexibility, not onlyto achieve increased affinity but also to enable better structuralconformation for internalization as compared to the parental clone andother mutant clones.

We confirmed that both the parental DMAb14-scFv-IT and mutantDMAb14-scFv-Mut-IT react with purified 3′-isoLM1 and 3′,6′-isoLD1,whereas D2C7-scFv-IT, which is an antibody to EGFRvIII and wild-typeEGFR, does not (FIG. 9). In addition, both DMAb14-scFv-Mut-IT andD2C7-scFv-IT show significant cytotoxicity against GBM tumor cells.Since these two immunotoxins target different tumor antigens,D2C7-scFv-IT is an excellent positive control for cytotoxicity in ourstudy.

The IC₅₀ values of the DMAb14-scFv-Mut-IT varied among the differentcell lines and xenografts tested (FIG. 10), probably due to differentnumbers of gangliosides on the cell surfaces. It is also possible that3′-isoLM1 and 3′,6′-isoLD1 are present at different proportions ondifferent cell types. Xenograft H2224 cells have been screened by ananti-ganglioside monoclonal antibody specific for 3′-isoLM1 (10) and3′,6′-isoLD1 (18), which revealed that 88% of H2224 cells were positivefor 3′-isoLM1 and 2% for 3′,6′-isoLD1. Therefore, 3′-isoLM1 is the mainganglioside expressed on H2224 tumor cells. We also determined that thexenograft H2224 cells have a higher ratio of 3′-isoLM1 to 3′,6′-isoLD1than H336 and D54 MG cells (data not shown). Since DMAb14-scFv-Mut-IThas a more potent effect on xenograft H2224 cells (IC₅₀ of 0.5 ng/mL)than on H336 cells (IC₅₀ of 2 ng/mL), DMAb14-scFv-Mut-IT may have agreater affinity for 3′-isoLM1 and greater benefit for patients with3′-isoLM1-dominant brain tumors.

The DMAb14-scFv parental and mutant immunotoxins differ only at thetargeted amino acid residues. Both show specificity and binding affinityto the ganglioside-positive cells; however, only the mutant IT displayscytotoxic activity. Therefore we sought to determine whether theimmunotoxins were differentially internalized by the cells. Recombinantimmunotoxins are chimeric proteins used for cancer therapy, consistingof a targeting moiety linked to the catalytic domains of a natural toxin(19). We utilized Pseudomonas exotoxin A (PE38), which is a potentbacterial toxin composed of three domains (20) that is able to entertumor cells and inhibit protein synthesis, eventually killing the cell.We found that DMAb14-scFv-Mut-IT is internalized significantly fasterthan the parental IT, with xenograft H2224 cells and H336 cellsinternalizing 94% and 98% of DMAb14-scFv-Mut-IT, respectively, within 4hours and internalizing no significant amount of the parental IT withinthe same time period (FIG. 12). Xenograft H2224 cells internalized themutant IT faster than H336 cells, which may explain whyDMAb14-scFv-Mut-IT is more potent in H2224 cells.

In summary, we developed an anti-ganglioside scFv immunotoxin with highcytotoxic activity and tumor specificity. We achieved this goal usingintrinsic mutational hot spots as targets for antibody affinitymaturation in vitro. The parental clone DMAb14-scFv-IT had the properaffinity but did not have any cytotoxic activity, whereas the selectedmutant, DMAb14-scFv-Mut-IT, showed a much higher affinity and increasedcytotoxic activity to ganglioside-positive cell lines and tumorxenograft cells. DMAb14-scFv-Mut-IT exhibited strong cytotoxic effectson the human glioblastoma xenograft cell line H2224 (IC₅₀=0.5 ng/mL) andno cytotoxic effects on the ganglioside-negative cell line D293,demonstrating the selectivity of the immunotoxin. Moreover, theincreased cytotoxic activity of the mutant immunotoxin could provide aclinical benefit with a low dose, which would decrease nonspecifictoxicities in patients. This study is, to the best of our knowledge, thefirst time a scFv recombinant immunotoxin targeting 3′-isoLM1 and3′,6′-isoLD1 gangliosides has been shown to have significantspecificity, high binding affinity, and dramatic cytotoxic activity intumor cells. Our findings strongly suggest that this immunotoxin hasgreat potential for clinical diagnosis and therapy for patients withbrain tumors and warrants further preclinical studies.

REFERENCES FOR EXAMPLE 6 ONLY

The disclosure of each reference cited is expressly incorporated herein.

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References for all but Example 6

The disclosure of each reference cited is expressly incorporated herein.

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1. An isolated antibody construct which comprises a V_(H)CDR2 accordingto SEQ ID NO:1 and a V_(L)CDR1 according to SEQ ID NO:2, wherein theantibody construct binds to both 3′-isoLM1 and 3′,6′-iso-LD1gangliosides.
 2. The isolated antibody construct of claim 1 which isattached to a cytotoxic moiety.
 3. The isolated antibody construct ofclaim 2 wherein the cytotoxic moiety is selected from the groupconsisting of biological toxins and radiologic toxic moieties.
 4. Theisolated antibody construct of claim 3 wherein the cytotoxic moiety isPseudomonas exotoxin A.
 5. The isolated antibody construct of claim 3wherein the cytotoxic moiety is PE38, a truncated form of Pseudomonasexotoxin A.
 6. An isolated nucleic acid molecule which encodes an scFvmolecule comprising a V_(H)CDR2 according to SEQ ID NO:1 and a V_(L)CDR1according to SEQ ID NO:2, wherein the scFv binds to both 3′-isoLM1 and3′,6′-iso-LD1 gangliosides.
 7. A host cell comprising the isolatednucleic acid molecule of claim
 6. 8. A method of making monoclonalantibody comprising: growing in culture medium the host cell of claim 7;and harvesting antibody construct from the culture medium or host cells.9. A monoclonal antibody which has a higher affinity for 3′,6′-isoLD1ganglioside than for 3′ isoLM1 ganglioside, and which does notspecifically bind to a ganglioside selected from the group consisting ofisoLA1, Fuc3′-isoLM1, 3′-LM1, 3′,8′-LD1, Gb3, GA1, GM2, GM1, GD3, GD2,GD1b, and GT1b.
 10. An IgG monoclonal antibody which binds to one ormore lacto-series gangliosides.
 11. The antibody of claim 10 which bindsto NeuAcα2-3Galβ1-3GlcNAc.
 12. The antibody of claim 10 which is an IgG₃type.
 13. The antibody of any of claims 9 to 12 which is attached to acytotoxic agent.
 14. The antibody of any of claims 9 to 12 wherein theantibody has an affinity constant for 3′ isoLM1 at binding equilibriumof at least 10⁷ (mol/L)⁻¹.
 15. The antibody of any of claims 9 to 12wherein the antibody has an affinity constant for 3′ isoLM1 at bindingequilibrium of at least 10⁸ (mol/L)⁻¹.
 16. The antibody of claim 10which does not bind to at least one ganglioside selected from the groupconsisting of: isoLA1, Fuc-3′-isoLM1, 3′-LM1, 3′,8′-LD1, LA1, Gb3, andGA1.
 17. The antibody of claim 9 or 10 which does not bind to: isoLA1,Fuc-3′-isoLM1, 3′-LM1, 3′,8′-LD1, LA1, Gb3, and GA1.
 18. A hybridomacell which produces the monoclonal antibody of any of claims 9-12.
 19. Amethod of making monoclonal antibody comprising: growing in culturemedium the hybridoma cells of claim 18; and harvesting monoclonalantibody from the culture medium or hybridoma cells.
 20. A method ofdetecting tumor cells in a tissue comprising: contacting a body samplewith an antibody according to any of claims 1 and 9 to 12; and detectingthe presence of antibody bound to the body sample.
 21. The method ofclaim 20 wherein the step of detecting employs a secondary antibody. 22.The method of claim 20 wherein the tumor is a glioblastoma.
 23. Themethod of claim 20 wherein the tumor is selected from the groupconsisting of breast, prostate, colon, renal cell, pancreas, melanoma,seminoma, germinoma, teratoma, and leiomyosarcoma.
 24. A method oftreating a tumor in a human comprising: administering to the human amonoclonal antibody or antibody construct according to any of claims 1and 9-12, wherein the monoclonal antibody or antibody construct isattached to a cytotoxic agent.
 25. The method of claim 24 wherein thetumor is a glioblastoma.
 26. The method of claim 24 wherein the tumor isselected from the group consisting of breast, prostate, colon, renalcell, pancreas, melanoma, seminoma, germinoma, teratoma, andleiomyosarcoma.